Anti-Pancreatic Cancer Antibodies

ABSTRACT

Described herein are compositions and methods of use of anti-pancreatic cancer antibodies or fragments thereof, such as murine, chimeric, humanized or human PAM4 antibodies. The subject antibodies show a number of novel and useful therapeutic characteristics, such as binding with high specificity to pancreatic and other cancers, but not to normal or benign pancreatic tissues and binding to a high percentage of early stage pancreatic cancers. In preferred embodiments, the antibodies bind to pancreatic cancer mucins. The antibodies and fragments are of use for the detection, diagnosis and/or treatment of cancer, such as pancreatic cancer. The antibodies, such as PAM4 antibodies, bind to a PAM4 antigen that shows unique cell and tissue distributions compared with other known antibodies such as CA19.9, DUPAN2, SPAN1, Nd2, B72.3, and Le a  and Le(y) antibodies that bind to the Lewis antigens.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.12/537,803 (now issued U.S. Pat. No. 8,491,896), filed Aug. 7, 2009,which was a continuation-in-part of U.S. patent application Ser. No.12/343,655 (now issued U.S. Pat. No. 7,993,626), filed Dec. 24, 2008,which was a continuation in part of U.S. patent application Ser. No.12/112,289 (now issued U.S. Pat. No. 7,563,433), filed Apr. 30, 2008,which was a continuation-in-part of U.S. patent application Ser. No.11/960,262 (now issued U.S. Pat. No. 7,597,876), filed Dec. 19, 2007,which claimed the benefit under 35 U.S.C. 119(e) of provisional U.S.Patent Application 60/884,521, filed Jan. 11, 2007.

Ser. No. 12/537,803 was a continuation-in-part of U.S. patentapplication Ser. No. 12/396,605 (now issued U.S. Pat. No. 7,858,070),filed Mar. 3, 2009, which was a divisional of U.S. patent applicationSer. No. 11/633,729 (now issued U.S. Pat. No. 7,527,787), filed Dec. 5,2006, which was a continuation-in-part of U.S. patent application Ser.Nos. 11/389,358 (now issued U.S. Pat. No. 7,550,143), filed Mar. 24,2006; 11/391,584 (now issued U.S. Pat. No. 7,521,056), filed Mar. 28,2006, and 11/478,021 (now issued U.S. Pat. No. 7,534,866), filed Jun.29, 2006, and which claimed the benefit under 35 U.S.C. 119(e) ofprovisional U.S. Patent Application Nos. 60/782,332, filed Mar. 14,2006; 60/728,292, filed Oct. 19, 2005; 60/751,196, filed Dec. 16, 2005;and No. 60/864,530, filed Nov. 6, 2006.

Ser. No. 12/537,803 was a continuation-in-part of U.S. patentapplication Ser. Nos. 11/925,408 (now issued U.S. Pat. No. 7,666,400),filed Oct. 26, 2007, and 12/418,877 (now issued U.S. Pat. No.7,906,118), filed Apr. 6, 2009, which claimed the benefit under 35U.S.C. 119(e) of U.S. Provisional Patent Application Ser. Nos.61/043,932, filed Apr. 10, 2008, 61/104,916, filed Oct. 13, 2008 and61/119,542, filed Dec. 3, 2008.

Ser. No. 12/537,803 was a continuation-in-part of U.S. patentapplication Ser. No. 11/849,791 (now abandoned), filed Sep. 4, 2007,which was a divisional of U.S. patent application Ser. No. 10/461,885(now issued U.S. Pat. No. 7,282,567) filed Jun. 16, 2003, which claimedthe benefit under 35 U.S.C. 119(e) of U.S. Provisional PatentApplication 60/388,314, filed Jun. 14, 2002. Ser. No. 12/537,803 claimsthe benefit under 35 U.S.C. 119(e) of Provisional U.S. PatentApplication Ser. Nos. 61/087,463, filed Aug. 8, 2008, and 61/144,227,filed Jan. 13, 2009. The text of each priority application cited aboveis incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This work was supported in part by NIH grant RO1-CA54425. The U.S.Government may have certain rights in this invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to murine, chimeric, humanized and humanantibodies and fragments thereof that bind with high selectivity andfrequency to pancreatic cancer cells and to a lesser extent to othercancer cells and not appreciably to normal pancreatic cells orpancreatitis. In preferred embodiments, the antibodies or fragments arePAM4 antibodies or fragments. The subject antibodies are of use incancer detection, diagnosis and therapy, particularly for pancreaticcancers. In particular embodiments, the subject antibodies are of usefor detection and/or diagnosis of the earliest stages of pancreaticcancer.

2. Related Art

Pancreatic cancer is a malignant growth of the pancreas that mainlyoccurs in the cells of the pancreatic ducts. This disease is the ninthmost common form of cancer, yet it is the fourth and fifth leading causeof cancer deaths in men and women, respectively. Cancer of the pancreasis almost always fatal, with a five-year survival rate that is less than3%.

The most common symptoms of pancreatic cancer include jaundice,abdominal pain, and weight loss, which, together with other presentingfactors, are nonspecific in nature. Thus, diagnosing pancreatic cancerat an early stage of tumor growth is often difficult and requiresextensive diagnostic work-up, often times including exploratory surgery.Endoscopic ultrasonogaphy and computed tomography are the bestnoninvasive means available today for diagnosis of pancreatic cancer.However, reliable detection of small tumors, as well as differentiationof pancreatic cancer from focal pancreatitis, is difficult. The vastmajority of patients with pancreatic cancer are presently diagnosed at alate stage when the tumor has already extended outside of the capsule toinvade surrounding organs and/or has metastasized extensively. Gold etal., Crit. Rev. Oncology/Hematology, 39:147-54 (2001). Late detection ofthe disease is common, and early pancreatic cancer diagnosis is rare inthe clinical setting.

Current treatment procedures available for pancreatic cancer have notled to a cure, nor to a substantially improved survival time. Surgicalresection has been the only modality that offers a chance at survival.However, due to a large tumor burden, only 10% to 25% of patients arecandidates for “curative resection.” For those patients undergoing asurgical treatment, the five-year survival rate is still poor, averagingonly about 10%.

Early detection and diagnosis of pancreatic cancer, as well asappropriate staging of the disease, would provide an increased survivaladvantage. A number of laboratories are proceeding on the development ofa diagnostic procedure based upon the release of a tumor-associatedmarker into the bloodstream as well as detection of the marker substancewithin biopsy specimens. The best tumor associated marker for pancreaticcancer has been the immunoassay for CA19.9. Elevated levels of thissialylated Le^(a) epitope structure were found in 70% of pancreaticcancer patients but were not found in any of the focal pancreatitisspecimens examined. However, CA19.9 levels were found to be elevated ina number of other malignant and benign conditions, so that currently theassay cannot be used for diagnosis. However, the assay is useful formonitoring, the continued increase in CA19.9 serum levels after surgerybeing indicative of a poor prognosis. Many other monoclonal antibodies(MAbs) have been reported with immunoassays for diagnosis in varyingstages of development. These include but are not limited to DUPAN2,SPAN1, B72.3, Ia3, and various anti-CEA (carcinoembryonic antigen, orCEACAM5) antibodies.

Antibodies, in particular MAbs and engineered antibodies or antibodyfragments, have been tested widely and shown to be of value in detectionand treatment of various human disorders, including cancers, autoimmunediseases, infectious diseases, inflammatory diseases, and cardiovasculardiseases [Filpula and McGuire, Exp. Opin. Ther. Patents (1999) 9:231-245]. The clinical utility of an antibody or an antibody-derivedagent is primarily dependent on its ability to bind to a specifictargeted antigen associated with a particular disorder. Selectivity isvaluable for delivering a diagnostic or therapeutic agent, such asdrugs, toxins, cytokines, hormones, hormone antagonists, enzymes, enzymeinhibitors, oligonucleotides, growth factors, radionuclides,angiogenesis inhibitors or metals, to a target location during thedetection and treatment phases of a human disorder, particularly if thediagnostic or therapeutic agent is toxic to normal tissue in the body.Radiolabeled antibodies have been used with some success in numerousmalignancies, including ovarian cancer, colon cancer, medullary thyroidcancer, and lymphomas. This technology may also prove useful forpancreatic cancer. However, previously reported antibodies againstpancreatic cancer antigens have not been successfully employed to datefor the effective therapy or early detection and/or diagnosis ofpancreatic cancer.

One suggested approach for delivering agents to a target site, referredto as direct targeting, is a technique designed to target specificantigens with antibodies carrying diagnostic or therapeutic agents. Inthe context of tumors, the direct targeting approach utilizes a labeledanti-tumor monospecific antibody that recognizes the target tumorthrough its antigens. The technique involves injecting the labeledmonospecific antibody into the patient and allowing the antibody tolocalize at the target tumor to obtain diagnostic or therapeuticbenefits. The unbound antibody clears the body.

Another suggested solution, referred to as the “Affinity EnhancementSystem” (AES), is a technique designed to overcome deficiencies ofdirect tumor targeting by antibodies carrying diagnostic or therapeuticagents [U.S. Pat. No. 5,256,395 (1993), Barbet et al., Cancer Biotherapy& Radiopharmaceuticals 14: 153-166 (1999)]. The AES utilizes a labeleddivalent hapten and an anti-tumor/anti-hapten bispecific antibody thatrecognizes both the target tumor and the labeled hapten. Haptens withhigher valency and antibodies with higher specificity may also beutilized for this procedure. The technique involves injecting theantibody into the patient and allowing it to localize at the targettumor. After a sufficient amount of time for the unbound antibody toclear from the blood stream, the labeled hapten is administered. Thehapten binds to the antibody-antigen complex located at the site of thetarget cell to obtain diagnostic or therapeutic benefits, while theunbound hapten rapidly clears from the body. Barbet mentions thepossibility that a bivalent hapten may crosslink with bispecificantibodies, when the latter are bound to the tumor surface. As a result,the labeled complex is more stable and stays at the tumor for a longerperiod of time.

There remains a need in the art for antibodies that exhibit highselectivity for pancreatic cancer and other types of cancers, comparedto normal pancreatic tissues and other normal tissues. Specifically,there remains a need for antibodies that perform as a useful diagnosticand/or therapeutic tool for pancreatic cancer, preferably at theearliest stages of the disease, and that exhibits enhanced uptake attargeted antigens, decreased binding to constituents in the blood ofhealthy individuals and thereby also optimal protection of normaltissues and cells from toxic therapeutic agents when these areconjugated to such antibodies. Use of such antibodies to detectpancreatic cancer-associated antigens in body fluids, particularlyblood, can enable improved earlier diagnosis of this disease, so long asit differentiates well from benign diseases, and can also be used formonitoring response to therapy and potentially also to enhance prognosisby indicating disease burden.

SUMMARY

In various embodiments, the present invention concerns antibodies,antigen-binding antibody fragments and fusion proteins that bind topancreatic cancer cells, with little or no binding to normal ornon-neoplastic pancreatic cells. Preferably, the antibodies bind to theearliest stages of pancreatic cancer, such as PanIN-1A and 1B andPanIN-2. More preferably, the antibodies bind to 80 to 90% or more ofhuman invasive pancreatic adenocarcinoma, intraductal papillary mucinousneoplasia, PanIN-1A, PanIN-1B and PanIN-2 lesions. Most preferably, theantibodies can distinguish between early stage pancreatic cancer andnon-malignant conditions such as pancreatitis. Such antibodies are ofparticular use for early detection of cancer and differential diagnosisbetween early stage pancreatic cancer and benign pancreatic conditions.In preferred embodiments, such antibodies are of use for in vivo or exvivo analysis of samples from individuals suspected of having earlystage pancreatic or certain other cancers.

The antibodies, antibody fragments or fusion proteins may be derived byimmunization and/or selection with mucin, and are preferably reactiveagainst mucin of pancreatic cancer. Accordingly, the antibodies,antibody fragments and fusion proteins preferably bind to an antigenassociated with pancreatic cancer cells. More preferably, theantibodies, antibody fragments or fusion proteins may bind to a mucinexpressed in pancreatic cancer, such as MUC-1 or MUC-5. In certainembodiments, the antibodies, antibody fragments or fusion proteins maybind to cells transfected with and expressing a MUC-1 antigen.

In alternative embodiments, the antibodies, antibody fragments or fusionproteins may bind to synthetic peptide sequences, for example to phagedisplay peptides. Exemplary peptides that may bind to theanti-pancreatic cancer antibodies include, but are not limited to,WTWNITKAYPLP (SEQ ID NO:29) and ACPEWWGTTC (SEQ ID NO:30). Suchsynthetic peptides may be linear or cyclic and may or may not competewith antibody binding to the endogenous pancreatic cancer antigen. Aminoacids in certain positions of the synthetic peptide sequences may beless critical for antibody binding than others. For example, in SEQ IDNO:29 the residues K, A and L at positions 7, 8 and 11 of the peptidesequence may be varied while still retaining antibody binding.Similarly, in SEQ ID NO:30 the threonine residues at positions 8 and 9of the sequence may be varied, although substitution of the threonine atposition 9 may significantly affect antibody binding to the peptide.

In other preferred embodiments, binding of the antibodies to a targetpancreatic cancer antigen is inhibited by treatment of the targetantigen with reagents such as dithiothreitol (DTT) and/or periodate.Thus, binding of the antibodies to a pancreatic cancer antigen may bedependent upon the presence of disulfide bonds and/or the glycosylationstate of the target antigen. In more preferred embodiments, the epitoperecognized by the subject antibodies is not cross-reactive with otherreported mucin-specific antibodies, such as the MA5 antibody, the CLH2-2antibody and/or the 45M1 antibody (see, e.g., Major et al., J HistochemCytochem. 35:139-48, 1987; Dion et al., Hybridoma 10:595-610, 1991).

The subject antibodies or fragments may be naked antibodies or fragmentsor preferably are conjugated to at least one therapeutic and/ordiagnostic agent for delivery of the agent to target tissues. Inalternative embodiments, the PAM4 antibodies or fragments may be part ofa bispecific fusion protein or antibody with a first binding site for atarget cell antigen and a second binding site for a hapten conjugated toa targetable construct. The targetable construct may in turn be attachedto at least one therapeutic and/or diagnostic agent, of use inpretargeting techniques.

In preferred embodiments, the subject antibody, antibody fragment orfusion protein comprises a murine, chimeric, humanized or human PAM4antibody or fragment. Such PAM4 antibodies or fragments preferablycomprise the CDR sequences of a murine PAM4 antibody, such as the lightchain variable region CDR sequences CDR1 (SASSSVSSSYLY, SEQ ID NO:1);CDR2 (STSNLAS, SEQ ID NO:2); and CDR3 (HQWNRYPYT, SEQ ID NO:3); and theheavy chain variable region CDR sequences CDR1 (SYVLH, SEQ ID NO:4);CDR2 (YINPYNDGTQYNEKFKG, SEQ ID NO:5) and CDR3 (GFGGSYGFAY, SEQ IDNO:6).

Particular embodiments may concern compositions and methods of use ofmurine PAM4 antibodies, preferably comprising murine PAM4 variableregion sequences as disclosed in SEQ ID NO:9 and SEQ ID NO:11. Suchmurine PAM4 antibodies or fragments may be of use in in vitro or ex vivodiagnostic techniques, such as immunohistochemical analysis of tissuesamples or immunoassay of body fluid samples from subjects suspected ofhaving pancreatic cancer or other types of cancer.

Other particular embodiments may concern compositions and methods of useof chimeric PAM4 antibodies, comprising murine variable region sequencesattached to human antibody constant region sequences. Preferably thechimeric PAM4 antibodies or fragments comprise the cPAM4 variable regionsequences shown in SEQ ID NO:12 (FIG. 2A) and SEQ ID NO:13 (FIG. 2B).Such chimeric antibodies are less immunogenic in humans than murineantibodies, while retaining the antigen-binding specificities of theparent murine antibody. Chimeric antibodies are well known in the artfor use in diagnostic and/or therapeutic treatment of cancer.

Still other particular embodiments may concern compositions, and methodsof use of humanized PAM4 antibodies or fragments thereof, comprising thecomplementarity-determining regions (CDRs) of a murine PAM4 MAb asdiscussed above and human antibody framework region (FR) and constantregion sequences. In a preferred embodiment, the FRs of the light andheavy chain variable regions of the humanized PAM4 antibody or fragmentthereof comprise at least one amino acid substituted from thecorresponding FRs of a murine PAM4 MAb. Still more preferred, thehumanized PAM4 antibody or fragment thereof may comprise at least aminoacid residue selected from amino acid residues 5, 27, 30, 38, 48, 66, 67and 69 of the murine PAM4 heavy chain variable region (SEQ ID NO:11)and/or at least one amino acid selected from amino acid residues 21, 47,59, 60, 85, 87 and 100 of the murine PAM4 light chain variable region(SEQ ID NO:9). Most preferably, the humanized PAM4 antibody or fragmentthereof comprises the hPAM4 VH amino acid sequence of SEQ ID NO:19 andthe hPAM4 Vκ amino acid sequence of SEQ ID NO:16.

In alternative embodiments, the anti-pancreatic cancer antibodies may bemurine, chimeric, humanized or human antibodies that bind to the sameantigenic determinant (epitope) as a chimeric PAM4 (cPAM4) antibody. Asdiscussed below, the cPAM4 antibody is one that comprises the lightchain variable region CDR sequences CDR1 (SASSSVSSSYLY, SEQ ID NO:1);CDR2 (STSNLAS, SEQ ID NO:2); and CDR3 (HQWNRYPYT, SEQ ID NO:3); and theheavy chain variable region CDR sequences CDR1 (SYVLH, SEQ ID NO:4);CDR2 (YINPYNDGTQYNEKFKG, SEQ ID NO:5) and CDR3 (GFGGSYGFAY, SEQ IDNO:6). Antibodies that bind to the same antigenic determinant may beidentified by a variety of techniques known in the art, such as bycompetitive binding studies using the cPAM4 antibody as the competingantibody and human pancreatic mucin as the target antigen. (See, e.g.,Example 1, paragraph [0214] below.) Antibodies that block (compete for)binding to human pancreatic mucin of a cPAM4 antibody are referred to ascross-blocking antibodies. Preferably, such cross-blocking antibodiesare prepared by immunization with extracts comprising human pancreaticcancer mucins.

Another embodiment is a cancer cell targeting diagnostic immunoconjugatecomprising an anti-pancreatic cancer antibody, antibody fragment orfusion protein that is bound to at least one diagnostic (or detection)agent.

Preferably, the diagnostic agent is selected from the group consistingof a radionuclide, a contrast agent, a fluorescent agent, achemiluminescent agent, a bioluminescent agent, a paramagnetic ion, anenzyme and a photoactive diagnostic agent. Still more preferred, thediagnostic agent is a radionuclide with an energy between 20 and 4,000keV or is a radionuclide selected from the group consisting of ¹¹⁰In,¹⁷⁷Lu, ¹⁸F, ⁵²Fe, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁸⁶Y, ⁹⁰Y, ⁸⁹Zr,^(94m)Tc, ⁹⁴Tc, ^(99m)Tc, ¹²⁰I, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹⁵⁴⁻¹⁵⁸Gd, ³²P,¹¹C, ¹³N, ¹⁵O, ¹⁸⁶Re, ¹⁸⁸Re, ⁵¹Mn, 52 mMn, ⁵⁵Co, ⁷²As, ⁷⁵Br, ⁷⁶Br,^(82m)Rb, ⁸³Sr, or other gamma-, beta-, or positron-emitters. In aparticularly preferred embodiment, the diagnostic radionuclide ¹⁸F isused for labeling and PET imaging, as described in the Examples below.The ¹⁸F may be attached to an antibody, antibody fragment or peptide bycomplexation to a metal, such as aluminum, and binding of the ¹⁸F-metalcomplex to a chelating moiety that is conjugated to a targeting protein,peptide or other molecule.

Also preferred, the diagnostic agent is a paramagnetic ion, such aschromium (III), manganese (II), iron (III), iron (II), cobalt (II),nickel (II), copper (II), neodymium (III), samarium (III), ytterbium(III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III),holmium (III) and erbium (III), or a radiopaque material, such asbarium, diatrizoate, ethiodized oil, gallium citrate, iocarmic acid,iocetamic acid, iodamide, iodipamide, iodoxamic acid, iogulamide,iohexyl, iopamidol, iopanoic acid, ioprocemic acid, iosefamic acid,ioseric acid, iosulamide meglumine, iosemetic acid, iotasul, iotetricacid, iothalamic acid, iotroxic acid, ioxaglic acid, ioxotrizoic acid,ipodate, meglumine, metrizamide, metrizoate, propyliodone, and thallouschloride.

In still other embodiments, the diagnostic agent is a fluorescentlabeling compound selected from the group consisting of fluoresceinisothiocyanate, rhodamine, phycoerytherin, phycocyanin, allophycocyanin,o-phthaldehyde and fluorescamine, a chemiluminescent labeling compoundselected from the group consisting of luminol, isoluminol, an aromaticacridinium ester, an imidazole, an acridinium salt and an oxalate ester,or a bioluminescent compound selected from the group consisting ofluciferin, luciferase and aequorin. In another embodiment, thediagnostic immunoconjugates are used in intraoperative, endoscopic, orintravascular tumor diagnosis.

Another embodiment is a cancer cell-targeting therapeuticimmunoconjugate comprising an antibody or fragment thereof or fusionprotein bound to at least one therapeutic agent. Preferably, thetherapeutic agent is selected from the group consisting of aradionuclide, an immunomodulator, a hormone, a hormone antagonist, anenzyme, an oligonucleotide such as an anti-sense oligonucleotide or asiRNA, an enzyme inhibitor, a photoactive therapeutic agent, a cytotoxicagent such as a drug or toxin, an angiogenesis inhibitor and apro-apoptotic agent. In embodiments where more than one therapeuticagent is used, the therapeutic agents may comprise multiple copies ofthe same therapeutic agent or else combinations of different therapeuticagents.

In one embodiment, an oligonucleotide, such as an antisense molecule orsiRNA inhibiting bcl-2 expression as described in U.S. Pat. No.5,734,033 (the Examples section of which is incorporated herein byreference), may be conjugated to, or form the therapeutic agent portionof an immunoconjugate or antibody fusion protein. Alternatively, theoligonucleotide may be administered concurrently or sequentially with anaked or conjugated anti-pancreatic cancer antibody or antibodyfragment, such as a PAM4 antibody. In a preferred embodiment, theoligonucleotide is an antisense oligonucleotide that is directed againstan oncogene or oncogene product, such as bcl-2, p53, ras or otherwell-known oncogenes.

In a preferred embodiment, the therapeutic agent is a cytotoxic agent,such as a drug or a toxin. Also preferred, the drug is selected from thegroup consisting of nitrogen mustards, ethylenimine derivatives, alkylsulfonates, nitrosoureas, gemcitabine, triazenes, folic acid analogs,anthracyclines, taxanes, COX-2 inhibitors, pyrimidine analogs, purineanalogs, antibiotics, enzyme inhibitors, epipodophyllotoxins, platinumcoordination complexes, vinca alkaloids, substituted ureas, methylhydrazine derivatives, adrenocortical suppressants, hormone antagonists,endostatin, taxols, camptothecins, SN-38, doxorubicins and theiranalogs, antimetabolites, alkylating agents, antimitotics,anti-angiogenic agents, tyrosine kinase inhibitors, mTOR inhibitors,heat shock protein (HSP90) inhibitors, proteosome inhibitors, HDACinhibitors, pro-apoptotic agents, methotrexate, CPT-11, and acombination thereof.

In another preferred embodiment, the therapeutic agent is a toxinselected from the group consisting of ricin, abrin, alpha toxin,saporin, ribonuclease (RNase), DNase I, Staphylococcal enterotoxin-A,pokeweed antiviral protein, gelonin, diphtheria toxin, Pseudomonasexotoxin, and Pseudomonas endotoxin and combinations thereof. Or animmunomodulator selected from the group consisting of a cytokine, a stemcell growth factor, a lymphotoxin, a hematopoietic factor, a colonystimulating factor (CSF), an interferon (IFN), a stem cell growthfactor, erythropoietin, thrombopoietin and a combinations thereof.

In other preferred embodiments, the therapeutic agent is a radionuclideselected from the group consisting of ¹¹¹In, ¹⁷⁷Lu, ²¹²Bi, ²¹³Bi, ²¹¹At,⁶²Cu, ⁶⁷Cu, ⁹⁰Y, ¹²⁵I, ¹³¹¹I, ³²P, ³³P, ⁴⁷Sc, ¹¹¹Ag, ⁶⁷Ga, ¹⁴²Pr, ¹⁵³Sm,¹⁶¹Tb, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re, ²¹²Pb, ²²³Ra, ²²⁵Ac, ⁵⁹Fe,⁷⁵Se, ⁷⁷As, ⁸⁹Sr, ⁹⁹Mo, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹⁴³Pr, ¹⁴⁹Pm, ¹⁶⁹Er, ¹⁹⁴Ir, ¹⁹⁸Au,¹⁹⁹Au, and ²¹¹Pb, and combinations thereof. Also preferred areradionuclides that substantially decay with Auger-emitting particles.For example, Co-58, Ga-67, Br-80m, Tc-99m, Rh-103m, Pt-109, In-111,Sb-119, 1-125, Ho-161, Os-189m and Ir-192. Decay energies of usefulbeta-particle-emitting nuclides are preferably <1,000 keV, morepreferably <100 keV, and most preferably <70 keV. Also preferred areradionuclides that substantially decay with generation ofalpha-particles. Such radionuclides include, but are not limited to:Dy-152, At-211, Bi-212, Ra-223, Rn-219, Po-215, Bi-211, Ac-225, Fr-221,At-217, Bi-213 and Fm-255. Decay energies of usefulalpha-particle-emitting radionuclides are preferably 2,000-10,000 keV,more preferably 3,000-8,000 keV, and most preferably 4,000-7,000 keV.Additional potential radioisotopes of use include ¹¹C, ¹³N, ¹⁵O, ⁷⁵Br,¹⁹⁸Au, ²²⁴Ac, ¹²⁶I, ¹³³I, ⁷⁷Br, ^(113m)In, ⁹⁵Ru, ⁹⁷Ru, ¹⁰³Ru, ¹⁰⁵Ru,¹⁰⁷Hg, ²⁰³Hg, ^(121m)Te, ^(122m)Te, ^(125m)Te, ¹⁶⁵Tm, ¹⁶⁷Tm, ¹⁶⁸Tm,¹⁹⁷Pt, ¹⁰⁹Pd, ¹⁰⁵Rh, ¹⁴²Pr, ¹⁴³Pr, ¹⁶¹Tb, ¹⁶⁶Ho, ¹⁹⁹Au, ⁵⁷Co, ⁵⁸Co,⁵¹Cr, ⁵⁹Fe, ⁷⁵Se, ²⁰¹Tl, ²²⁵Ac, ⁷⁶Br, ¹⁶⁹Yb, and the like. In otherembodiments the therapeutic agent is a photoactive therapeutic agentselected from the group consisting of chromogens and dyes.

Alternatively, the therapeutic agent is an enzyme selected from thegroup consisting of malate dehydrogenase, staphylococcal nuclease,delta-V-steroid isomerase, yeast alcohol dehydrogenase,alpha-glycerophosphate dehydrogenase, triose phosphate isomerase,horseradish peroxidase, alkaline phosphatase, asparaginase, glucoseoxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase. Such enzymes may be used, for example, incombination with prodrugs that are administered in relatively non-toxicform and converted at the target site by the enzyme into a cytotoxicagent. In other alternatives, a drug may be converted into less toxicform by endogenous enzymes in the subject but may be reconverted into acytotoxic form by the therapeutic enzyme.

Also contemplated are multivalent, multispecific antibodies or fragmentsthereof comprising at least one binding site having an affinity toward aPAM4 target antigen and one or more hapten binding sites having affinitytowards hapten molecules. Preferably, the antibody or fragment thereofis a chimeric, humanized or fully human antibody or fragment thereof.The hapten molecule may be conjugated to a targetable construct fordelivery of one or more therapeutic and/or diagnostic agents. In certainpreferred embodiments, the multivalent antibodies or fragments thereofmay be prepared by the dock-and-lock (DNL) technique, as described inthe Examples below. An exemplary DNL construct incorporating hPAM4antibody fragments is designated TF10, as described below.

Also contemplated is a bispecific antibody or fragment thereofcomprising at least one binding site with an affinity toward a PAM4target antigen and at least one binding site with an affinity toward atargetable construct/conjugates selected from the group consisting of:

DOTA-D-Asp-D-Lys(HSG)-D-Asp-D-Lys(HSG)-NH₂ (IMP 271);DOTA-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH₂ (IMP 277);DOTA-D-Tyr-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH₂ (IMP 288);DOTA-D-Ala-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH₂ (IMP 0281);NOTA-ITC benzyl-D-Ala-D-Lys(HSG)-D-Tyr-D- Lys(HSG)-NH₂ (IMP 449);NODA-Ga-D-Ala-D-Lys(HSG)-D-Tyr-D-Lys(HSG)- NH₂ (IMP 460);NOTA-D-Ala-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH₂ (IMP 461);NOTA-D-Asp-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH₂ (IMP 462);NOTA-D-Ala-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH₂ (IMP 465);C-NETA-succinyl-D-Lys(HSG)-D-Tyr-D-Lys(HSG)- NH₂ (IMP 467);S-NETA-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH₂ (IMP 469); andL-NETA-succinyl-D-Lys(HSG)-D-Tyr-D-Lys(HSG)- NH₂ (IMP 470)that is capable of carrying at least one diagnostic and/or therapeuticagent. Other targetable constructs suitable for use are disclosed, forexample, in U.S. Pat. Nos. 6,576,746; 6,962,702; 7,052,872; 7,138,103;7,172,751 and 7,405,320 and U.S. patent application Ser. No. 12/112,289,the Examples section of each of which is incorporated herein byreference.

Other embodiments concern fusion proteins or fragments thereofcomprising at least two anti-pancreatic cancer antibodies and fragmentsthereof as described herein. Alternatively, the fusion protein orfragment thereof may comprise at least one first anti-pancreatic cancerantibody or fragment thereof and at least one second MAb or fragmentthereof. Preferably, the second MAb binds to a tumor-associated antigen,for example selected from the group consisting of CA19.9, DUPAN2, SPAN1,Nd2, B72.3, CC49, CEA (CEACAM5), CEACAM6, Le^(a), the Lewis antigenLe(y), CSAp, insulin-like growth factor (ILGF), epithelialglycoprotein-1 (EGP-1), epithelial glycoprotein-2 (EGP-2), CD-80,placental growth factor (P1GF), carbonic anhydrase IX, tenascin, IL-6,HLA-DR, CD40, CD74 (e.g., milatuzumab), CD138 (syndecan-1), MUC-1,MUC-2, MUC-3, MUC-4, MUC-5ac, MUC-16, MUC-17, TAG-72, EGFR,platelet-derived growth factor (PDGF), angiogenesis factors (e.g., VEGFand P1GF), products of oncogenes (e.g., bcl-2, Kras, p53), cMET,HER2/neu, and antigens associated with gastric cancer and colorectalcancer. The antibody fusion protein or fragments thereof may furthercomprise at least one diagnostic and/or therapeutic agent.

Also described herein are DNA sequences comprising a nucleic acidencoding an anti-pancreatic cancer antibody, fusion protein,multispecific antibody, bispecific antibody or fragment thereof asdescribed herein. Other embodiments concern expression vectors and/orhost cells comprising the antibody encoding DNA sequences. In certainpreferred embodiments, the host cell may be an Sp2/0 cell linetransformed with a mutant Bcl-2 gene, for example with a triple mutantBcl-2 gene (T69E, S70E, S87E), that has been adapted to celltransformation and growth in serum free medium. (See, e.g., U.S. patentapplication Ser. Nos. 11/187,863 (now issued U.S. Pat. No. 7,531,327),filed Jul. 25, 2005; 11/487,215 (now issued U.S. Pat. No. 7,537,930),filed Jul. 14, 2006; and 11/877,728, filed Oct. 24, 2007, the Examplessection of each of which is incorporated herein by reference.)

Another embodiment concerns methods of delivering a diagnostic ortherapeutic agent, or a combination thereof, to a target comprising (i)providing a composition that comprises an anti-pancreatic cancerantibody or fragment, such as a PAM4 antibody or fragment, conjugated toat least one diagnostic and/or therapeutic agent and (ii) administeringto a subject in need thereof the diagnostic or therapeutic conjugate ofany one of the antibodies, antibody fragments or fusion proteins claimedherein.

Also contemplated is a method of delivering a diagnostic agent, atherapeutic agent, or a combination thereof to a target, comprising: (a)administering to a subject any one of the multivalent, multispecific orbispecific antibodies or fragments thereof that have an affinity towarda PAM4 antigen and comprise one or more hapten binding sites; (b)waiting a sufficient amount of time for antibody that does not bind tothe PAM4 antigen to clear the subject's blood stream; and (c)administering to said subject a carrier molecule comprising a diagnosticagent, a therapeutic agent, or a combination thereof, that binds to abinding site of the antibody. Preferably, the carrier molecule binds tomore than one binding site of the antibody.

Described herein is a method for diagnosing or treating cancer,comprising: (a) administering to a subject any one of the multivalent,multispecific antibodies or fragments thereof claimed herein that havean affinity toward a PAM4 antigen and comprise one or more haptenbinding sites; (b) waiting a sufficient amount of time for an amount ofthe non-bound antibody to clear the subject's blood stream; and (c)administering to said subject a carrier molecule comprising a diagnosticagent, a therapeutic agent, or a combination thereof, that binds to abinding site of the antibody. In a preferred embodiment the cancer ispancreatic cancer. Also preferred, the method can be used forintraoperative identification of diseased tissues, endoscopicidentification of diseased tissues, or intravascular identification ofdiseased tissues.

Another embodiment is a method of treating a malignancy in a subjectcomprising administering to said subject a therapeutically effectiveamount of an antibody or fragment thereof that binds to a PAM4 antigen,optionally conjugated to at least one therapeutic agent. The antibody orfragment thereof may alternatively be a naked antibody or fragmentthereof. In more preferred embodiments, the antibody or fragment isadministered either before, simultaneously with, or after administrationof another therapeutic agent as described above.

Contemplated herein is a method of diagnosing a malignancy in a subject,particularly a pancreatic cancer, comprising (a) administering to saidsubject a diagnostic conjugate comprising an antibody or fragmentthereof that binds to a PAM4 antigen, wherein said MAb or fragmentthereof is conjugated to at least one diagnostic agent, and (b)detecting the presence of labeled antibody bound to pancreatic cancercells or other malignant cells, wherein binding of the antibody isdiagnostic for the presence of pancreatic cancer or another malignancy.In preferred embodiments, the antibody or fragment binds to pancreaticcancer and not to normal pancreatic tissue, pancreatitis or othernon-malignant conditions. In less preferred embodiments, the antibody orfragment binds at a significantly higher level to cancer cells than tonon-malignant cells, allowing differential diagnosis of cancer fromnon-malignant conditions. In a most preferred embodiment, the diagnosticagent may be an F-18 labeled molecule that is detected by PET imaging.

In more preferred embodiments, the use of anti-pancreatic cancerantibodies, such as the hPAM4 antibody, allows the detection and/ordiagnosis of pancreatic cancer with high specificity and sensitivity atthe earliest stages of malignant disease. Preferably, the diagnosticantibody or fragment is capable of labeling at least 70%, morepreferably at least 80%, more preferably at least 90%, more preferablyat least 95%, most preferably about 100% of well differentiated,moderately differentiated and poorly differentiated pancreatic cancerand 90% or more of invasive pancreatic adenocarcinomas. Theanti-pancreatic cancer antibody of use is preferably capable ofdetecting 85% or more of PanIN-1A, PanIN-1B, PanIN-2, IPMN and MCNprecursor lesions. Most preferably, immunoassays using theanti-pancreatic cancer antibody are capable of detecting 89% or more oftotal PanIN, 86% or more of IPMN, and 92% or more of MCN.

An alternative embodiment is a method of detecting PAM4 antigen and/ordiagnosing pancreatic cancer in an individual by in vitro analysis ofblood, plasma or serum samples. Preferably, the sample is subjected toan organic phase extraction, using an organic solvent such as butanol,before it is processed for immunodetection using an anti-pancreaticcancer antibody, such as a PAM4 antibody. Following organic phaseextraction, aqueous phase is analyzed for the presence of PAM4 antigenin the sample, using any of a variety of immunoassay techniques known inthe art, such as ELISA, sandwich immunoassay, solid phase RIA, andsimilar techniques.

Another embodiment is a method of treating a cancer cell in a subjectcomprising administering to said subject a composition comprising anaked antibody or fragment thereof or a naked antibody fusion protein orfragment thereof that binds to a PAM4 antigen. Preferably, the methodfurther comprises administering a second naked antibody or fragmentthereof selected from the group consisting of CA19.9, DUPAN2, SPAN1,Nd2, B72.3, CC49, anti-CEA, anti-CEACAM6, anti-EGP-1, anti-EGP-2,anti-Le^(a), antibodies defined by the Lewis antigen Le(y), andantibodies against CSAp, MUC-1, MUC-2, MUC-3, MUC-4, MUC-5ac, MUC-16,MUC-17, TAG-72, EGFR, CD40, HLA-DR, CD74, CD138, angiogenesis factors(e.g., VEGF and placenta-like growth factor (P1GF), insulin-like growthfactor (ILGF), tenascin, platelet-derived growth factor, IL-6, productsof oncogenes, cMET, and HER2/neu.

Still other embodiments concern a method of diagnosing a malignancy in asubject comprising (i) performing an in vitro diagnosis assay on aspecimen from said subject with a composition comprising an antibody orfragment thereof that binds to a PAM4 antigen; and (ii) detecting thepresence of antibody or fragment bound to malignant cells in thespecimen. Preferably, the malignancy is a cancer. Still preferred, thecancer is pancreatic cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A. Variable region cDNA sequence (SEQ ID NO:8) and the deducedamino acid (SEQ ID NO:9) sequence of the murine PAM4 Vk. Amino acidsequences encoded by the corresponding DNA sequences are given asone-letter codes below the nucleotide sequence. Numbering of thenucleotide sequence is on the right side. The amino acid residues in theCDR regions are shown in bold and underlined. Kabat's Ig moleculenumbering is used for amino acid residues as shown by the numberingabove the amino acid residues. The amino acid residues numbered by aletter are the insertion residues defined by Kabat numbering scheme. Theinsertion residues have the same preceding digits as that of theprevious residue.

FIG. 1B. Variable region cDNA sequence (SEQ ID NO:10) and amino acidsequence (SEQ ID NO:11) of the murine PAM4 VH. Amino acid sequencesencoded by the corresponding DNA sequences are given as one-letter codesbelow the nucleotide sequence. Numbering of the nucleotide sequence ison the right side. The amino acid residues in the CDR regions are shownin bold and underlined. Kabat's Ig molecule numbering is used for aminoacid residues as shown by the numbering above the amino acid residues.The amino acid residues numbered by a letter are the insertion residuesdefined by Kabat numbering scheme. The insertion residues have the samepreceding digits as that of the previous residue. For example, residues82, 82A, 82B, and 82C in FIG. 1B are indicated as 82, A, B, and C,respectively.

FIG. 2A. Amino acid sequence (SEQ ID NO:12) of the chimeric PAM4 (cPAM4)Vk. The sequences are given as one letter codes. The amino acid residuesin the CDR regions are shown in bold and underlined. Kabat's Ig moleculenumber scheme is used to number the residues.

FIG. 2B. Amino acid sequence (SEQ ID NO:13) of the cPAM4 VH. Thesequences are given as one letter codes. The amino acid residues in theCDR regions are shown in bold and underlined. Kabat's Ig molecule numberscheme is used to number the residues.

FIG. 3A. Alignment of the Vκ amino acid sequences of the human antibodyWalker (SEQ ID NO:14) with PAM4 (SEQ ID NO:9) and hPAM4 (SEQ ID NO:16).Dots indicate the residues of PAM4 that are identical to thecorresponding residues of the human or humanized antibodies. Boxedregions represent the CDR regions. Both N- and C-terminal residues(underlined) of hPAM4 are fixed by the staging vectors used. Kabat's Igmolecule number scheme is used to number the residues.

FIG. 3B. Alignment of the VH amino acid sequences of the human antibodyWil2 (FR1-3) (SEQ ID NO:17) and NEWM (FR4) (SEQ ID NO:28) with PAM4 (SEQID NO:11) and hPAM4 (SEQ ID NO:19). Dots indicate the residues of PAM4that are identical to the corresponding residues of the human orhumanized antibodies. Boxed regions represent the CDR regions. Both N-and C-terminal residues (underlined) of hPAM4 are fixed by the stagingvectors used. Kabat's Ig molecule number scheme is used to number theresidues.

FIG. 4A. DNA (SEQ ID NO:15) and amino acid (SEQ ID NO:16) sequences ofthe humanized PAM4 (hPAM4) Vκ. Numbering of the nucleotide sequence ison the right side. Amino acid sequences encoded by the corresponding DNAsequences are given as one-letter codes. The amino acid residues in theCDR regions are shown in bold and underlined. Kabat's Ig moleculenumbering scheme is used for amino acid residues.

FIG. 4B. DNA (SEQ ID NO:18) and amino acid (SEQ ID NO:19) sequences ofthe hPAM4 VH. Numbering of the nucleotide sequence is on the right side.Amino acid sequences encoded by the corresponding DNA sequences aregiven as one-letter codes. The amino acid residues in the CDR regionsare shown in bold and underlined. Kabat's Ig molecule numbering schemeis used for amino acid residues.

FIG. 5. Binding activity of humanized PAM4 antibody, hPAM4, as comparedto the chimeric PAM4, cPAM4. hPAM4 is shown by diamonds and cPAM4 isshown by closed circles. Results indicate comparable binding activity ofthe hPAM4 antibody and cPAM4 when competing with ¹²⁵I-cPAM4 binding toCaPan1 antigens.

FIG. 6. PET/CT fusion images for a patient with inoperable metastaticpancreatic cancer treated with fractionated ⁹⁰Y-hPAM4 plus gemcitabine,before therapy (left side) and post-therapy (right side). The circleindicates the location of the primary lesion, which shows a significantdecrease in PET/CT intensity following therapy.

FIG. 7. 3D PET images for a patient with inoperable metastaticpancreatic cancer treated with fractionated ⁹⁰Y-hPAM4 plus gemcitabine,before therapy (left side) and post-therapy (right side). Arrows pointto the locations of the primary lesion (on right) and metastases (onleft), each of which shows a significant decrease in PET image intensityafter therapy with radiolabeled hPAM4 plus gemcitabine.

FIG. 8. In vivo imaging of tumors using an ¹¹¹In-labeled diHSG peptide(IMP 288) with or without pretargeting TF10 bispecific anti-pancreaticcancer mucin antibody. FIG. 8A—mice showing location of tumors (arrow).FIG. 8B shows the detected tumors with ¹¹¹In-labeled IMP 288 in thepresence (above) or absence (below) of TF10 bispecific antibody.

FIG. 9. Exemplary binding curves for TF10, PAM4-IgG, PAM4-F(ab′)₂ and amonovalent bsPAM4 chemical conjugate (PAM4-Fab′ x anti-DTPA-Fab′).Binding to target mucin antigen was measured by ELISA assay.

FIG. 10. Immunoscintigraphy of CaPan1 human pancreatic cancer xenografts(˜0.25 g). (A) An image of mice that were injected with bispecific TF 10(80 μg, 5.07×10⁻¹⁰ mol) followed 16 h later by administration of¹¹¹In-IMP-288 (30 μCi, 5.07×10⁻¹¹ mol). The image was taken 3 h later.The intensity of the image background was increased to match theintensity of the image obtained when ¹¹¹In-IMP-288 was administeredalone (30 μCi, 5.07×10⁻¹¹ mol). (B) No targeting was observed in micegiven ¹¹¹In-IMP-288 alone. (C) An image of mice that were given¹¹¹In-DOTA-PAM4-IgG (20 μCi, 50 μg) with imaging done 24 h later.Although tumors are visible, considerable background activity is stillpresent at this time point.

FIG. 11A. Extended biodistribution of ¹¹¹In-DOTA-PAM4-IgG (20 μCi, 50μg) and TF10-pretargeted ¹¹¹In-IMP-288 (80 μg, 5.07×10⁻¹⁰ mol TF10followed 16 h later with 30 5.07×10⁻¹¹ mol ¹¹¹In-IMP-288) in nude micebearing CaPan1 human pancreatic cancer xenografts (mean tumorweight+/−SD, 0.28+/−0.21 and 0.10+/−0.06 g for the pretargeting and IgGgroups of animals, respectively). FIG. 11A shows percent of initial doseper gram of tissue in tumor with PAM4 IgG (open circles), blood withPAM4 IgG (open squares), tumor with pretargeted peptide (closed circles)and blood with pretargeted peptide (closed squares).

FIG. 11B. Extended biodistribution of ¹¹¹In-DOTA-PAM4-IgG (20 μCi, 50μg) and TF10-pretargeted ¹¹¹In-IMP-288 (80 μg, 5.07×10⁻¹⁰ mol TF10followed 16 h later with 30 μCi, 5.07×10⁻¹¹ mol ¹¹¹In-IMP-288) in nudemice bearing CaPan1 human pancreatic cancer xenografts (mean tumorweight+/−SD, 0.28+/−0.21 and 0.10+/−0.06 g for the pretargeting and IgGgroups of animals, respectively). FIG. 11B shows percent of initial doseper gram of tissue in liver with PAM4 IgG (open triangles), kidney withPAM4 IgG (open diamonds), liver with pretargeted peptide (closedtriangles) and kidney with pretargeted peptide (closed diamonds).

FIG. 11C. Extended biodistribution of ¹¹¹In-DOTA-PAM4-IgG (20 μCi, 50μg) and TF10-pretargeted ¹¹¹In-IMP-288 (80 μg, 5.07×10⁻¹⁰ mol TF10followed 16 h later with 30 μCi, 5.07×10⁻¹¹ mol ¹¹¹In-IMP-288) in nudemice bearing CaPan1 human pancreatic cancer xenografts (mean tumorweight+/−SD, 0.28+/−0.21 and 0.10+/−0.06 g for the pretargeting and IgGgroups of animals, respectively). FIG. 11C shows microcuries per gram oftissue in tumor with PAM4 IgG (open circles), blood with PAM4 IgG (opensquares), tumor with pretargeted peptide (closed circles) and blood withpretargeted peptide (closed squares).

FIG. 11D. Extended biodistribution of ¹¹¹In-DOTA-PAM4-IgG (20 μCi, 50μg) and TF10-pretargeted ¹¹¹In-IMP-288 (80 μg, 5.07×10¹⁰ mol TF10followed 16 h later with 30 μCi, 5.07×10⁻¹¹ mol in nude mice bearingCaPan1 human pancreatic cancer xenografts (mean tumor weight+/−SD,0.28+/−0.21 and 0.10+/−0.06 g for the pretargeting and IgG groups ofanimals, respectively). FIG. 11D shows microcuries per gram of tissue inliver with PAM4 IgG (open triangles), kidney with PAM4 IgG (opendiamonds), liver with pretargeted peptide (closed triangles) and kidneywith pretargeted peptide (closed diamonds).

FIG. 12. Therapeutic activity of a single treatment of established (˜0.4cm³) CaPan1 tumors with 0.15 mCi of ⁹⁰Y-hPAM4 IgG, or 0.25 or 0.50 mCiof TF10-pretargeted ⁹⁰Y-IMP-288.

FIG. 13. Effect of gemcitabine potentiation of PT-RAIT therapy.

FIG. 14. Effect of combination of cetuximab with gemcitabine andPT-RAIT.

FIG. 15. Differential diagnosis of pancreatic cancer using PAM4-basedimmunoassay. The red line shows the cutoff level selected for a positiveresult, based on ROC analysis.

FIG. 16. Frequency distribution of PAM4 antigen in patient sera fromhealthy volunteers and individuals with varying stages of pancreaticcancer.

FIG. 17. ROC curve for PAM4 serum immunoassay.

DETAILED DESCRIPTION Definitions

Unless otherwise specified, “a” or “an” means one or more.

As used herein, “about” means plus or minus 10%. For example, “about100” would include any number between 90 and 110.

The term “substantially less” means at least 90%, more preferably 95%,more preferably 98%, more preferably 99%, more preferably 99.9% less.

As described herein, the term “PAM4 antibody” includes murine, chimeric,humanized and human PAM4 antibodies. In preferred embodiments, the PAM4antibody or antigen-binding fragment thereof comprises the CDR sequencesof SEQ ID NO:1 to SEQ ID NO:6.

As used herein, a “PAM4 antigen” is an antigen bound by a PAM4 antibody.In preferred embodiments, treatment of PAM4 antigen with DTT orperiodate inhibits or prevents binding of the PAM4 antibody. In morepreferred embodiments, the PAM4 antigen is an epitope of a mucinexpressed by a pancreatic cancer cell, such as MUC-1 or MUC-5.

As used herein, an “anti-pancreatic cancer antibody” is an antibody thatexhibits the same diagnostic, therapeutic and binding characteristics asthe PAM4 antibody. In preferred embodiments, the “anti-pancreatic cancerantibody” binds to the same epitope as the PAM4 antibody.

A “non-endocrine pancreatic cancer” generally refers to cancers arisingfrom the exocrine pancreatic glands. The term excludes pancreaticinsulinomas and includes pancreatic carcinoma, pancreaticadenocarcinoma, adenosquamous carcinoma, squamous cell carcinoma andgiant cell carcinoma and precursor lesions such as pancreaticintra-epithelial neoplasia (PanIN), mucinous cyst neoplasms (MCN) andintrapancreatic mucinous neoplasms (IPMN), which are neoplastic but notyet malignant. The terms “pancreatic cancer” and “non-endocrinepancreatic cancer” are used interchangeably herein.

An antibody, as described herein, refers to a full-length (i.e.,naturally occurring or formed by normal immunoglobulin gene fragmentrecombinatorial processes) immunoglobulin molecule (e.g., an IgGantibody) or an immunologically active (i.e., specifically binding)portion of an immunoglobulin molecule, like an antibody fragment.

An antibody fragment is a portion of an antibody such as F(ab′)₂, Fab′,Fab, Fv, sFv and the like. Regardless of structure, an antibody fragmentbinds with the same antigen that is recognized by the full-lengthantibody. The term “antibody fragment” also includes isolated fragmentsconsisting of the variable regions of antibodies, such as the “Fv”fragments consisting of the variable regions of the heavy and lightchains and recombinant single chain polypeptide molecules in which lightand heavy variable regions are connected by a peptide linker (“scFvproteins”).

A naked antibody is an antibody or fragment thereof that is notconjugated to a therapeutic or diagnostic agent. Generally, the Fcportion of the antibody molecule provides effector functions, such ascomplement-mediated cytotoxicity (CDC) and ADCC (antibody-dependentcellular cytotoxicity), which set mechanisms into action that may resultin cell lysis. However, the Fc portion may not be required fortherapeutic function, with other mechanisms, such as signaling-inducedapoptosis, coming into play. Naked antibodies include both polyclonaland monoclonal antibodies, as well as fusion proteins and certainrecombinant antibodies, such as chimeric, humanized or human antibodies.

A chimeric antibody is a recombinant protein that contains the variabledomains including the complementarity determining regions (CDRs) of anantibody derived from one species, preferably a rodent antibody, whilethe constant domains of the antibody molecule are derived from those ofa human antibody. For veterinary applications, the constant domains ofthe chimeric antibody may be derived from that of other species, such asa cat or dog.

A humanized antibody is a recombinant protein in which the CDRs from anantibody from one species; e.g., a rodent antibody, are transferred fromthe heavy and light variable chains of the rodent antibody into humanheavy and light variable domains (e.g., framework region sequences). Theconstant domains of the antibody molecule are derived from those of ahuman antibody. In certain embodiments, a limited number of frameworkregion amino acid residues from the parent (rodent) antibody may besubstituted into the human antibody framework region sequences.

A human antibody is, e.g., an antibody obtained from transgenic micethat have been “engineered” to produce specific human antibodies inresponse to antigenic challenge. In this technique, elements of thehuman heavy and light chain loci are introduced into strains of micederived from embryonic stem cell lines that contain targeted disruptionsof the endogenous murine heavy chain and light chain loci. Thetransgenic mice can synthesize human antibodies specific for particularantigens, and the mice can be used to produce human antibody-secretinghybridomas. Methods for obtaining human antibodies from transgenic miceare described by Green et al., Nature Genet. 7:13 (1994), Lonberg etal., Nature 368:856 (1994), and Taylor et al., Int. Immun. 6:579 (1994).A fully human antibody also can be constructed by genetic or chromosomaltransfection methods, as well as phage display technology, all of whichare known in the art. See for example, McCafferty et al., Nature348:552-553 (1990) for the production of human antibodies and fragmentsthereof in vitro, from immunoglobulin variable domain gene repertoiresfrom unimmunized donors. In this technique, antibody variable domaingenes are cloned in-frame into either a major or minor coat protein geneof a filamentous bacteriophage, and displayed as functional antibodyfragments on the surface of the phage particle. Because the filamentousparticle contains a single-stranded DNA copy of the phage genome,selections based on the functional properties of the antibody alsoresult in selection of the gene encoding the antibody exhibiting thoseproperties. In this way, the phage mimics some of the properties of theB cell. Phage display can be performed in a variety of formats, forreview, see e.g. Johnson and Chiswell, Current Opiniion in StructuralBiology 3:5564-571 (1993). Human antibodies may also be generated by invitro activated B cells. See U.S. Pat. Nos. 5,567,610 and 5,229,275, theExamples section of which are incorporated herein by reference.

A therapeutic agent is a compound, molecule or atom which isadministered separately, concurrently or sequentially with an antibodymoiety or conjugated to an antibody moiety, i.e., antibody or antibodyfragment, or a subfragment, and is useful in the treatment of a disease.Examples of therapeutic agents include antibodies, antibody fragments,drugs, toxins, nucleases, hormones, immunomodulators, pro-apoptoticagents, anti-angiogenic agents, boron compounds, photoactive agents ordyes and radioisotopes. Therapeutic agents of use are described in moredetail below.

A diagnostic agent is a molecule, atom or other detectable moiety whichmay be administered conjugated to an antibody moiety or targetableconstruct and is useful in detecting or diagnosing a disease by locatingcells containing the target antigen. Useful diagnostic agents include,but are not limited to, radioisotopes, dyes (such as with thebiotin-streptavidin complex), contrast agents, fluorescent compounds ormolecules and enhancing agents (e.g., paramagnetic ions) for magneticresonance imaging (MRI) or positron emission tomography (PET) scanning.Preferably, the diagnostic agents are selected from the group consistingof radioisotopes, enhancing agents for use in magnetic resonanceimaging, and fluorescent compounds. In order to load an antibodycomponent with radioactive metals or paramagnetic ions, it may benecessary to react it with a reagent having a long tail to which areattached a multiplicity of chelating groups for binding the ions. Such atail can be a polymer such as a polylysine, polysaccharide, or otherderivatized or derivatizable chain having pendant groups to which can bebound chelating groups such as, e.g., ethylenediaminetetraacetic acid(EDTA), diethylenetriaminepentaacetic acid (DTPA), DOTA, NOTA, NETA,porphyrins, polyamines, crown ethers, bis-thiosemicarbazones,polyoximes, and like groups known to be useful for this purpose.Chelates are coupled to the antibodies using standard chemistries. Thechelate is normally linked to the antibody by a group which enablesformation of a bond to the molecule with minimal loss ofimmunoreactivity and minimal aggregation and/or internal cross-linking.Other, more unusual, methods and reagents for conjugating chelates toantibodies are disclosed in U.S. Pat. No. 4,824,659, the Examplessection of which is incorporated herein by reference. Particularlyuseful metal-chelate combinations include 2-benzyl-DTPA and itsmonomethyl and cyclohexyl analogs, used with diagnostic isotopes in thegeneral energy range of 60 to 4,000 keV, such as ¹²⁵I, ¹³¹I, ¹²³I, ¹²⁴I,⁶²Cu, ⁶⁴Cu, ¹⁸F, ¹¹¹In, ⁶⁷Ga, ⁶⁸Ga, ^(99m)Tc, ^(94m)Tc, ¹¹C, ¹³N, ¹⁵O,⁷⁶Br, for radioimaging. The same chelates, when complexed withnon-radioactive metals, such as manganese, iron and gadolinium areuseful for MRI, when used along with the antibodies of the invention.Macrocyclic chelates such as NOTA(1,4,7-triazacyclononane-N,N′,N″-triacetic acid), DOTA(1,4,7,10-tetraazacyclododecanetetraacetic acid), and TETA(p-bromoacetamido-benzyl-tetraethylaminetetraacetic acid) are of usewith a variety of metals and radiometals, most particularly withradionuclides of gallium, yttrium and copper, respectively. Suchmetal-chelate complexes can be made very stable by tailoring the ringsize to the metal of interest. Other ring-type chelates such asmacrocyclic polyethers, which are of interest for stably bindingnuclides, such as ²²³Ra for radioimmunotherapy (RAIT) are encompassed bythe invention. More recently, techniques of general utility for labelingvirtually any molecule with an ¹⁸F atom of use in PET imaging have beendescribed in U.S. patent application Ser. No. 12/112,289 (now issuedU.S. Pat. No. 7,563,433), the Examples section of which is incorporatedherein by reference.

An immunoconjugate is an antibody, antibody fragment or fusion proteinconjugated to at least one therapeutic and/or diagnostic agent. Thediagnostic agent can comprise a radionuclide or non-radionuclide, acontrast agent (such as for magnetic resonance imaging, computedtomography or ultrasound), and the radionuclide can be a gamma-, beta-,alpha-, Auger electron-, or positron-emitting isotope. The therapeuticagent may be any agent of use to treat a disease state such as cancer,described in more detail below.

An expression vector is a DNA molecule comprising a gene that isexpressed in a host cell. Typically, gene expression is placed under thecontrol of certain regulatory elements, including constitutive orinducible promoters, tissue-specific regulatory elements and enhancers.Such a gene is said to be “operably linked to” the regulatory elements.

A recombinant host may be any prokaryotic or eukaryotic cell thatcontains either a cloning vector or expression vector. This term alsoincludes those prokaryotic or eukaryotic cells, as well as transgenicanimals, that have been genetically engineered to contain the clonedgene(s) in the chromosome or genome of the host cell. Suitable mammalianhost cells include myeloma cells, such as SP2/0 cells, and NS0 cells, aswell as Chinese Hamster Ovary (CHO) cells, hybridoma cell lines andother mammalian host cell useful for expressing antibodies. Alsoparticularly useful to express mAbs and other fusion proteins are Sp2/0cells transfected with an apoptosis inhibitor, such as a Bcl-EEE gene,and adapted to grow and be further transfected in serum free conditions,as described in U11/187,863 (now issued U.S. Pat. No. 7,531,327), filedJul. 25, 2005; 11/487,215 (now issued U.S. Pat. No. 7,537,930), filedJul. 14, 2006; and 11/877,728, filed Oct. 24, 2007, the Examples sectionof each of which is incorporated herein by reference.)

As used herein, the term antibody fusion protein is a recombinantlyproduced antigen-binding molecule in which two or more of the same ordifferent natural antibody, single-chain antibody or antibody fragmentswith the same or different specificities are linked. Valency of thefusion protein indicates the total number of binding arms or sites thefusion protein has to an antigen or epitope; i.e., monovalent, bivalent,trivalent or multivalent. The multivalency of the antibody fusionprotein means that it can take advantage of multiple interactions inbinding to an antigen, thus increasing the avidity of binding to theantigen. Specificity indicates how many antigens or epitopes an antibodyfusion protein is able to bind; i.e., monospecific, bispecific,trispecific, multispecific. Using these definitions, a natural antibody,e.g., an IgG, is bivalent because it has two binding arms but ismonospecific because it binds to one antigen. Monospecific, multivalentfusion proteins have more than one binding site for an epitope but onlybind with the same or different epitopes on the same antigen, forexample a diabody with two binding sites reactive with the same antigen.The fusion protein may comprise a multivalent or multispecificcombination of different antibody components or multiple copies of thesame antibody component. The fusion protein may additionally comprise atherapeutic agent. Examples of therapeutic agents suitable for suchfusion proteins include immunomodulators (“antibody-immunomodulatorfusion protein”) and toxins (“antibody-toxin fusion protein”). Onepreferred toxin comprises a ribonuclease (RNase), preferably arecombinant RNase.

A multispecific antibody is an antibody that can bind simultaneously toat least two targets that are of different structure, e.g., twodifferent antigens, two different epitopes on the same antigen, or ahapten and/or an antigen or epitope. Multispecific, multivalentantibodies are constructs that have more than one binding site, and thebinding sites are of different specificity.

A bispecific antibody is an antibody that can bind simultaneously to twodifferent targets. Bispecific antibodies (bsAb) and bispecific antibodyfragments (bsFab) may have at least one arm that specifically binds to,for example, a tumor-associated antigen and at least one other arm thatspecifically binds to a targetable conjugate that bears a therapeutic ordiagnostic agent. A variety of bispecific fusion proteins can beproduced using molecular engineering.

PAM4 Antibodies

Various embodiments of the invention concern antibodies that react withvery high selectivity with pancreatic cancer as opposed to normal orbenign pancreatic tissues. The anti-pancreatic cancer antibodies andfragments thereof are preferably raised against a crude mucinpreparation from a tumor of the human pancreas, although partiallypurified or even purified mucins may be utilized. A non-limiting exampleof such antibodies is the PAM4 antibody.

The murine PAM4 (mPAM4) antibody was developed by employing pancreaticcancer mucin derived from the xenografted RIP-1 human pancreaticcarcinoma as immunogen. (Gold et al., Int. J. Cancer, 57:204-210, 1994.)As discussed below, antibody cross-reactivity and immunohistochemicalstaining studies indicate that the PAM4 antibody recognizes a unique andnovel epitope on a target pancreatic cancer antigen. Immunohistochemicalstaining studies, such as those described in Example 2, have shown thatthe PAM4 MAb binds to an antigen expressed by breast, pancreas and othercancer cells, with limited binding to normal human tissue; however, thehighest expression is usually by pancreatic cancer cells. Thus, the PAM4antibodies are relatively specific to pancreatic cancer andpreferentially bind pancreatic cancer cells. The PAM4 antibody isreactive with a target epitope which can be internalized. This epitopeis expressed primarily by antigens associated with pancreatic cancer andnot with focal pancreatitis or normal pancreatic tissue. Binding of PAM4antibody to the PAM4 epitope is inhibited by treatment of the antigenwith DTT or periodate. Localization and therapy studies using aradiolabeled PAM4 MAb in animal models have demonstrated tumor targetingand therapeutic efficacy.

The PAM4 antibodies bind to PAM4 antigen, which is expressed by manyorgans and tumor types, but is preferentially expressed in pancreaticcancer cells. Studies with a PAM4 MAb, such as in Example 3, indicatethat the antibody exhibits several important properties, which make it agood candidate for clinical diagnostic and therapeutic applications. ThePAM4 antigen provides a useful target for diagnosis and therapy ofpancreatic and other cancers. The PAM4 antibody apparently recognizes anepitope of a pancreatic cancer antigen that is distinct from theepitopes recognized by non-PAM4 anti-pancreatic cancer antibodies(CA19.9, DUPAN2, SPAN1, Nd2, CEACAM5, B72.3, anti-Le^(a), and otheranti-Lewis antigens).

Antibodies suitable for use in combination or conjunction with PAM4antibodies include, for example, the antibodies CA19.9, DUPAN2, SPAN1,Nd2, B72.3, CC49, anti-CEA, anti-CEACAM6, anti-Le^(a), anti-HLA-DR,anti-CD40, anti-CD74, anti-CD138, and antibodies defined by the Lewisantigen Le(y), or antibodies against colon-specific antigen-p (CSAp),MUC-1, MUC-2, MUC-3, MUC-4, MUC-5ac, MUC-16, MUC-17, EGP-1, EGP-2,HER2/neu, EGFR, angiogenesis factors (e.g., VEGF and P1GF), insulin-likegrowth factor (ILGF), tenascin, platelet-derived growth factor, andIL-6, as well as products of oncogenes (bcl-2, Kras, p53), cMET, andantibodies against tumor necrosis substances, such as described inpatents by Epstein et al. (U.S. Pat. Nos. 6,071,491, 6,017,514,5,019,368 and 5,882,626). Such antibodies would be useful forcomplementing PAM4 antibody immunodetection and immunotherapy methods.These and other therapeutic agents could act synergistically withanti-pancreatic cancer antibodies, such as PAM4 antibody, whenadministered before, together with or after administration of PAM4antibody.

In therapeutic applications, antibodies that are agonistic orantagonistic to immunomodulators involved in effector cell functionagainst tumor cells could also be useful in combination with PAM4antibodies alone or in combination with other tumor-associatedantibodies, one example being antibodies against CD40. Todryk et al., J.Immunol Methods, 248:139-147 (2001); Turner et al., J. Immunol,166:89-94 (2001). Also of use are antibodies against markers or productsof oncogenes (e.g., bcl-2, Kras, p53, cMET), or antibodies againstangiogenesis factors, such as VEGFR and placenta-like growth factor(P1GF).

The availability of another PAM4-like antibody that binds to a differentepitope of the PAM4 antigen is important for the development of adouble-determinant enzyme-linked immunosorbent assay (ELISA), of use fordetecting a PAM4 antigen in clinical samples. ELISA experiments aredescribed in Example 1 and 5.

The murine, chimeric, humanized and fully human PAM4 antibodies andfragments thereof described herein are exemplary of anti-pancreaticcancer antibodies of use for diagnostic and/or therapeutic methods. TheExamples below disclose a preferred embodiment of the construction anduse of a humanized PMA4 antibody. Because non-human monoclonalantibodies can be recognized by the human host as a foreign protein, andrepeated injections can lead to harmful hypersensitivity reactions,humanization of a murine antibody sequences can reduce the adverseimmune response that patients may experience. For murine-basedmonoclonal antibodies, this is often referred to as a Human Anti-MouseAntibody (HAMA) response. Preferably some human residues in theframework regions of the humanized anti-pancreatic cancer antibody orfragments thereof are replaced by their murine counterparts. It is alsopreferred that a combination of framework sequences from two differenthuman antibodies is used for VH. The constant domains of the antibodymolecule are derived from those of a human antibody.

Another preferred embodiment is a human anti-pancreatic cancer antibody,such as a human PAM4 antibody. A human antibody is an antibody obtained,for example, from transgenic mice that have been “engineered” to producespecific human antibodies in response to antigenic challenge. In thistechnique, elements of the human heavy and light chain locus areintroduced into strains of mice derived from embryonic stem cell linesthat contain targeted disruptions of the endogenous heavy chain andlight chain loci. The transgenic mice can synthesize human antibodiesspecific for human antigens, and the mice can be used to produce humanantibody-secreting hybridomas. Methods for obtaining human antibodiesfrom transgenic mice are described by Green et al., Nature Genet. 7:13(1994), Lonberg et al., Nature 368:856 (1994), and Taylor et al., Int.Immun. 6:579 (1994).

A fully human antibody also can be constructed by genetic or chromosomaltransfection methods, as well as phage display technology, all of whichare known in the art. See for example, McCafferty et al., Nature348:552-553 (1990) for the production of human antibodies and fragmentsthereof in vitro, from immunoglobulin variable domain gene repertoiresfrom unimmunized donors. In this technique, antibody variable domaingenes are cloned in-frame into either a major or minor coat protein geneof a filamentous bacteriophage, and displayed as functional antibodyfragments on the surface of the phage particle. Because the filamentousparticle contains a single-stranded DNA copy of the phage genome,selections based on the functional properties of the antibody alsoresult in selection of the gene encoding the antibody exhibiting thoseproperties. In this way, the phage mimics some of the properties of theB cell. Phage display can be performed in a variety of formats, fortheir review, see e.g. Johnson and Chiswell, Current Opinion inStructural Biology 3:5564-571 (1993).

Antibody Preparation

Monoclonal antibodies for specific antigens may be obtained by methodsknown to those skilled in the art. See, for example, Kohler andMilstein, Nature 256: 495 (1975), and Coligan et al. (eds.), CURRENTPROTOCOLS IN IMMUNOLOGY, VOL. 1, pages 2.5.1-2.6.7 (John Wiley & Sons1991) (hereinafter “Coligan”). Briefly, anti-pancreatic cancer MAbs canbe obtained by injecting mice with a composition comprising mucins frompancreatic cancer, such as the PAM4 antigen, verifying the presence ofantibody production by removing a serum sample, removing the spleen toobtain B-lymphocytes, fusing the B-lymphocytes with myeloma cells toproduce hybridomas, cloning the hybridomas, selecting positive cloneswhich produce antibodies to PAM4 antigen, culturing the clones thatproduce antibodies to PAM4 antigen, and isolating anti-pancreatic cancerantibodies from the hybridoma cultures.

After the initial raising of antibodies to the immunogen, the antibodiescan be sequenced and subsequently prepared by recombinant techniques toproduce chimeric or humanized antibodies. Chimerization of murineantibodies and antibody fragments are well known to those skilled in theart. The use of antibody components derived from chimerized monoclonalantibodies reduces potential problems associated with the immunogenicityof murine constant regions.

General techniques for cloning murine immunoglobulin variable domainsare described, for example, by the publication of Orlandi et al., ProcNat'l Acad. Sci. USA 86: 3833 (1989), incorporated herein by reference.In general, the V_(K) (variable light chain) and V_(H) (variable heavychain) sequences for murine antibodies can be obtained by a variety ofmolecular cloning procedures, such as RT-PCR, 5′-RACE, and cDNA libraryscreening. Specifically, the V_(H) and V_(K) sequences of the murinePAM4 MAb were cloned by PCR amplification from the hybridoma cells byRT-PCR, and their sequences determined by DNA sequencing. To confirmtheir authenticity, the cloned V_(K) and V_(H) genes can be expressed incell culture as a chimeric Ab as described by Orlandi et al., (ProcNatl. Acad. Sci., USA, 86: 3833, 1989).

In a preferred embodiment, a chimerized PAM4 antibody or antibodyfragment comprises the complementarity-determining regions (CDRs) andframework regions (FR) of a murine PAM4 MAb and the light and heavychain constant regions of a human antibody, wherein the CDRs of thelight chain variable region of the chimerized PAM4 comprises CDR1comprising an amino acid sequence of SASSSVSSSYLY (SEQ ID NO:1); CDR2comprising an amino acid sequence of STSNLAS (SEQ ID NO:2); and CDR3comprising an amino acid sequence of HQWNRYPYT (SEQ ID NO:3); and theCDRs of the heavy chain variable region of the chimerized PAM4 MAbcomprises CDR1 comprising an amino acid sequence of SYVLH (SEQ ID NO:4);CDR2 comprising an amino acid sequence of YINPYNDGTQYNEKFKG (SEQ IDNO:5) and CDR3 comprising an amino acid sequence of GFGGSYGFAY (SEQ IDNO:6). The use of antibody components derived from chimerized monoclonalantibodies reduces potential problems associated with the immunogenicityof murine constant regions.

Humanization of murine antibodies and antibody fragments is also wellknown to those skilled in the art. Techniques for producing humanizedMAbs are disclosed, for example, by Carter et al., Proc Nat'l Acad. Sci.USA 89: 4285 (1992), Singer et al., J. Immun. 150: 2844 (1992), Mountainet al. Biotechnol Genet Eng Rev. 10:1 (1992), and Coligan at pages10.19.1-10.19.11, each of which is incorporated herein by reference. Forexample, humanized monoclonal antibodies may be produced by transferringmurine complementary determining regions from heavy and light variablechains of the mouse immunoglobulin into a human variable domain, andthen, substituting human residues in the framework regions of the murinecounterparts. The use of human framework region sequences, in additionto human constant region sequences, further reduces the chance ofinducing HAMA reactions.

Based on the PAM4 variable region sequences, obtained as describedabove, a humanized PAM4 antibody can be designed and constructed asdescribed by Leung et al. (Mol Immunol. 32: 1413 (1995)), incorporatedherein by reference. Example 1 describes the humanization processutilized for construction of the hPAM4 MAb.

The nucleotide sequences of the primers used to prepare the hPAM4antibodies are discussed in Example 1, below. In a preferred embodiment,a humanized PAM4 antibody or antibody fragment comprises the light andheavy chain CDR sequences (SEQ ID NO:1 to SEQ ID NO:6) disclosed above.Also preferred, the FRs of the light and heavy chain variable regions ofthe humanized antibody comprise at least one amino acid substituted fromsaid corresponding FRs of the murine PAM4 MAb.

A fully human antibody, e.g., human PAM4 can be obtained from atransgenic non-human animal. See, e.g., Mendez et al., Nature Genetics,15: 146-156 (1997); U.S. Pat. No. 5,633,425. For example, a humanantibody can be recovered from a transgenic mouse possessing humanimmunoglobulin loci. The mouse humoral immune system is humanized byinactivating the endogenous immunoglobulin genes and introducing humanimmunoglobulin loci. The human immunoglobulin loci are exceedinglycomplex and comprise a large number of discrete segments which togetheroccupy almost 0.2% of the human genome. To ensure that transgenic miceare capable of producing adequate repertoires of antibodies, largeportions of human heavy- and light-chain loci must be introduced intothe mouse genome. This is accomplished in a stepwise process beginningwith the formation of yeast artificial chromosomes (YACs) containingeither human heavy- or light-chain immunoglobulin loci in germlineconfiguration. Since each insert is approximately 1 Mb in size, YACconstruction requires homologous recombination of overlapping fragmentsof the immunoglobulin loci. The two YACs, one containing the heavy-chainloci and one containing the light-chain loci, are introduced separatelyinto mice via fusion of YAC-containing yeast spheroblasts with mouseembryonic stem cells. Embryonic stem cell clones are then microinjectedinto mouse blastocysts. Resulting chimeric males are screened for theirability to transmit the YAC through their germline and are bred withmice deficient in murine antibody production. Breeding the twotransgenic strains, one containing the human heavy-chain loci and theother containing the human light-chain loci, creates progeny whichproduce human antibodies in response to immunization.

Antibodies can be produced by cell culture techniques using methodsknown in the art. In one example transfectoma cultures are adapted toserum-free medium. For production of humanized antibody, cells may begrown as a 500 ml culture in roller bottles using HSFM. Cultures arecentrifuged and the supernatant filtered through a 0.2 μm membrane. Thefiltered medium is passed through a protein-A column (1×3 cm) at a flowrate of 1 ml/min. The resin is then washed with about 10 column volumesof PBS and protein A-bound antibody is eluted from the column with 0.1 Mglycine buffer (pH 3.5) containing 10 mM EDTA. Fractions of 1.0 ml arecollected in tubes containing 10 μl of 3 M Tris (pH 8.6), and proteinconcentrations determined from the absorbance at 280/260 nm. Peakfractions are pooled, dialyzed against PBS, and the antibodyconcentrated, for example, with a Centricon 30 filter (Amicon, Beverly,Mass.). The antibody concentration is determined by ELISA and itsconcentration adjusted to about 1 mg/ml using PBS. Sodium azide, 0.01%(w/v), is conveniently added to the sample as preservative.

Antibodies can be isolated and purified from hybridoma cultures by avariety of well-established techniques. Such isolation techniquesinclude affinity chromatography with Protein-A Sepharose, size-exclusionchromatography, and ion-exchange chromatography. See, for example,Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3. Also, see Baines etal., “Purification of Immunoglobulin G (IgG),” in METHODS IN MOLECULARBIOLOGY, VOL. 10, pages 79-104 (The Humana Press, Inc. 1992).

Anti-pancreatic cancer MAbs can be characterized by a variety oftechniques that are well-known to those of skill in the art. Forexample, the ability of an antibody to bind to the PAM4 antigen can beverified using an indirect enzyme immunoassay, flow cytometry analysis,ELISA or Western blot analysis.

Antibody Fragments

Antibody fragments are antigen binding portions of an antibody, such asF(ab′)₂, Fab′, F(ab)₂, Fab, Fv, sFv, scFv and the like. Antibodyfragments which recognize specific epitopes can be generated by knowntechniques. F(ab′)₂ fragments, for example, can be produced by pepsindigestion of the antibody molecule. These and other methods aredescribed, for example, by Goldenberg, U.S. Pat. Nos. 4,036,945 and4,331,647 and references contained therein. Also, see Nisonoff et al.,Arch Biochem. Biophys. 89: 230 (1960); Porter, Biochem. J. 73: 119(1959), Edelman et al., in METHODS IN ENZYMOLOGY VOL. 1, page 422(Academic Press 1967), and Coligan at pages 2.8.1-2.8.10 and2.10.-2.10.4. Alternatively, Fab′ expression libraries can beconstructed (Huse et al., 1989, Science, 246:1274-1281) to allow rapidand easy identification of monoclonal Fab′ fragments with the desiredspecificity.

A single chain Fv molecule (scFv) comprises a VL domain and a VH domain.The VL and VH domains associate to form a target binding site. These twodomains are further covalently linked by a peptide linker (L). A scFvmolecule is denoted as either VL-L-VH if the VL domain is the N-terminalpart of the scFv molecule, or as VH-L-VL if the VH domain is theN-terminal part of the scFv molecule. Methods for making scFv moleculesand designing suitable peptide linkers are described in U.S. Pat. No.4,704,692, U.S. Pat. No. 4,946,778, R. Raag and M. Whitlow, “SingleChain Fvs.” FASEB Vol 9:73-80 (1995) and R. E. Bird and B. W. Walker,Single Chain Antibody Variable Regions, TIBTECH, Vol 9: 132-137 (1991).

Other antibody fragments, for example single domain antibody fragments,are known in the art and may be used in the claimed constructs. Singledomain antibodies (VHH) may be obtained, for example, from camels,alpacas or llamas by standard immunization techniques. (See, e.g.,Muyldermans et al., TIBS 26:230-235, 2001; Yau et al., J Immunol Methods281:161-75, 2003; Maass et al., J Immunol Methods 324:13-25, 2007). TheVHH may have potent antigen-binding capacity and can interact with novelepitopes that are inacessible to conventional VH-VL pairs. (Muyldermanset al., 2001). Alpaca serum IgG contains about 50% camelid heavy chainonly IgG antibodies (HCAbs) (Maass et al., 2007). Alpacas may beimmunized with known antigens, such as TNF-α, and VHHs can be isolatedthat bind to and neutralize the target antigen (Maass et al., 2007). PCRprimers that amplify virtually all alpaca VHH coding sequences have beenidentified and may be used to construct alpaca VHH phage displaylibraries, which can be used for antibody fragment isolation by standardbiopanning techniques well known in the art (Maass et al., 2007).

An antibody fragment can also be prepared by proteolytic hydrolysis of afull-length antibody or by expression in E. coli or another host of theDNA coding for the fragment. An antibody fragment can be obtained bypepsin or papain digestion of full-length antibodies by conventionalmethods. For example, an antibody fragment can be produced by enzymaticcleavage of antibodies with pepsin to provide an approximate 100 Kdfragment denoted F(ab′)₂. This fragment can be further cleaved using athiol reducing agent, and optionally a blocking group for the sulfhydrylgroups resulting from cleavage of disulfide linkages, to produce anapproximate 50 Kd Fab′ monovalent fragment. Alternatively, an enzymaticcleavage using papain produces two monovalent Fab fragments and an Fcfragment directly.

Another form of antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). A CDR is a segment of thevariable region of an antibody that is complementary in structure to theepitope to which the antibody binds and is more variable than the restof the variable region. Accordingly, a CDR is sometimes referred to as ahypervariable region. A variable region comprises three CDRs. CDRpeptides can be obtained by constructing genes encoding the CDR of anantibody of interest. Such genes are prepared, for example, by using thepolymerase chain reaction (PCR) to synthesize the variable region fromRNA of antibody-producing cells. See, for example, Larrick et al.,Methods: A Companion to Methods in Enzymology 2: 106 (1991);Courtenay-Luck, “Genetic Manipulation of Monoclonal Antibodies,” inMONOCLONAL ANTIBODIES: PRODUCTION, ENGINEERING AND CLINICAL APPLICATION,Ritter et al. (eds.), pages 166-179 (Cambridge University Press 1995);and Ward et al., “Genetic Manipulation and Expression of Antibodies,” inMONOCLONAL ANTIBODIES: PRINCIPLES AND APPLICATIONS, Birch et al.,(eds.), pages 137-185 (Wiley-Liss, Inc. 1995).

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

Antibody Fusion Proteins

Fusion proteins comprising the anti-pancreatic cancer antibodies ofinterest can be prepared by a variety of conventional procedures,ranging from glutaraldehyde linkage to more specific linkages betweenfunctional groups. The antibodies and/or antibody fragments thatcomprise the fusion proteins described herein are preferably covalentlybound to one another, directly or through a linker moiety, through oneor more functional groups on the antibody or fragment, e.g., amine,carboxyl, phenyl, thiol, or hydroxyl groups. Various conventionallinkers in addition to glutaraldehyde can be used, e.g., diisocyanates,diiosothiocyanates, bis(hydroxysuccinimide)esters, carbodiimides,maleimidehydroxy succinimide esters, and the like.

A simple method for producing fusion proteins is to mix the antibodiesor fragments in the presence of glutaraldehyde. The initial Schiff baselinkages can be stabilized, e.g., by borohydride reduction to secondaryamines. A diiosothiocyanate or carbodiimide can be used in place ofglutaraldehyde as a non-site-specific linker. In one embodiment, anantibody fusion protein comprises an anti-pancreatic cancer MAb, orfragment thereof, wherein the MAb binds to the PAM4 antigen. This fusionprotein and fragments thereof preferentially bind pancreatic cancercells. This monovalent, monospecific MAb is useful for direct targetingof an antigen, where the MAb is attached to a therapeutic agent, adiagnostic agent, or a combination thereof, and the protein isadministered directly to a patient.

The fusion proteins may instead comprise at least two anti-pancreaticcancer MAbs that bind to distinct epitopes of the PAM4 antigen. Forexample, the MAbs can produce antigen specific diabodies, triabodies andtetrabodies, which are multivalent but monospecific to the PAM4 antigen.The non-covalent association of two or more scFv molecules can formfunctional diabodies, triabodies and tetrabodies. Monospecific diabodiesare homodimers of the same scFv, where each scFv comprises the VH domainfrom the selected antibody connected by a short linker to the VL domainof the same antibody. A diabody is a bivalent dimer formed by thenon-covalent association of two scFvs, yielding two Fv binding sites. Atriabody results from the formation of a trivalent trimer of threescFvs, yielding three binding sites, and a tetrabody is a tetravalenttetramer of four scFvs, resulting in four binding sites. Severalmonospecific diabodies have been made using an expression vector thatcontains a recombinant gene construct comprising VH1-linker-VL1. SeeHolliger et al., Proc Natl. Acad. Sci USA 90: 6444-6448 (1993); Atwellet al., Molecular Immunology 33: 1301-1302 (1996); Holliger et al.,Nature Biotechnology 15: 632-631 (1997); Helfrich et al., Int J Cancer76: 232-239 (1998); Kipriyanov et al., Int J Cancer 77: 763-772 (1998);Holiger et al., Cancer Research 59: 2909-2916 (1999)). Methods ofconstructing scFvs are disclosed in U.S. Pat. No. 4,946,778 (1990) andU.S. Pat. No. 5,132,405 (1992), the Examples section of each of which isincorporated herein by reference. Methods of producing multivalent,monospecific antibodies based on scFv are disclosed in U.S. Pat. No.5,837,242 (1998), U.S. Pat. No. 5,844,094 (1998) and WO-98/44001 (1998),the Examples section of each of which is incorporated herein byreference. The multivalent, monospecific antibody fusion protein bindsto two or more of the same type of epitopes that can be situated on thesame antigen or on separate antigens. The increased valency allows foradditional interaction, increased affinity, and longer residence times.These antibody fusion proteins can be utilized in direct targetingsystems, where the antibody fusion protein is conjugated to atherapeutic agent, a diagnostic agent, or a combination thereof, andadministered directly to a patient in need thereof.

A preferred embodiment is a multivalent, multispecific antibody orfragment thereof comprising one or more antigen binding sites having anaffinity toward a PAM4 target epitope and one or more additional bindingsites for other epitopes associated with pancreatic cancer. This fusionprotein is multispecific because it binds at least two differentepitopes, which can reside on the same or different antigens. Forexample, the fusion protein may comprise more than one antigen bindingsite, the first with an affinity toward a PAM4 antigen epitope and thesecond with an affinity toward another target antigen such as TAG-72 orCEA. Another example is a bispecific antibody fusion protein which maycomprise a CA19.9 MAb (or fragment thereof) and a PAM4 MAb (or fragmentthereof). Such a fusion protein will have an affinity toward CA19.9 aswell as the PAM4 antigen. The antibody fusion proteins and fragmentsthereof can be utilized in direct targeting systems, where the antibodyfusion protein is conjugated to a therapeutic agent, a diagnostic agent,or a combination thereof, and administered directly to a patient in needthereof.

Another preferred embodiment is a multivalent, multispecific antibodycomprising at least one binding site having affinity toward a PAM4target epitope and at least one hapten binding site having affinitytowards hapten molecules. For example, a bispecific fusion protein maycomprise the 679 MAb (or fragment thereof) and the PAM4 MAb (or fragmentthereof). The monoclonal 679 antibody binds with high affinity tomolecules containing the tri-peptide moiety histamine succinyl glycyl(HSG). Such a bispecific PAM4 antibody fusion protein can be prepared,for example, by obtaining a F(ab′)₂ fragment from 679, as describedabove. The interchain disulfide bridges of the 679 F(ab′)₂ fragment aregently reduced with DTT, taking care to avoid light-heavy chain linkage,to form Fab′-SH fragments. The SH group(s) is (are) activated with anexcess of bis-maleimide linker(1,1′-(methylenedi-4,1-phenylene)b-is-malemide). The PAM4 MAb isconverted to Fab′-SH and then reacted with the activated 679 Fab′-SHfragment to obtain a bispecific antibody fusion protein. Bispecificantibody fusion proteins such as this one can be utilized in affinityenhancing systems, where the target antigen is pretargeted with thefusion protein and is subsequently targeted with a diagnostic ortherapeutic agent attached to a carrier moiety (targetable construct)containing one or more HSG haptens. In alternative preferredembodiments, a DNL-based hPAM4-679 construct, such as TF10, may beprepared and used as described in the Examples below.

Bispecific antibodies can be made by a variety of conventional methods,e.g., disulfide cleavage and reformation of mixtures of whole IgG or,preferably F(ab′)₂ fragments, fusions of more than one hybridoma to formpolyomas that produce antibodies having more than one specificity, andby genetic engineering. Bispecific antibody fusion proteins have beenprepared by oxidative cleavage of Fab′ fragments resulting fromreductive cleavage of different antibodies. This is advantageouslycarried out by mixing two different F(ab′)₂ fragments produced by pepsindigestion of two different antibodies, reductive cleavage to form amixture of Fab′ fragments, followed by oxidative reformation of thedisulfide linkages to produce a mixture of F(ab′)₂ fragments includingbispecific antibody fusion proteins containing a Fab′ portion specificto each of the original epitopes. General techniques for the preparationof antibody fusion proteins may be found, for example, in Nisonoff etal., Arch Biochem Biophys. 93: 470 (1961), Hmmerling et al., J Exp Med.128: 1461 (1968), and U.S. Pat. No. 4,331,647.

More selective linkage can be achieved by using a heterobifunctionallinker such as maleimidehydroxysuccinimide ester. Reaction of the esterwith an antibody or fragment will derivatize amine groups on theantibody or fragment, and the derivative can then be reacted with, e.g.,an antibody Fab fragment having free sulfhydryl groups (or, a largerfragment or intact antibody with sulfhydryl groups appended thereto by,e.g., Traut's Reagent). Such a linker is less likely to crosslink groupsin the same antibody and improves the selectivity of the linkage.

It is advantageous to link the antibodies or fragments at sites remotefrom the antigen-binding sites. This can be accomplished by, e.g.,linkage to cleaved interchain sulfydryl groups, as noted above. Anothermethod involves reacting an antibody having an oxidized carbohydrateportion with another antibody that has at lease one free amine function.This results in an initial Schiff base linkage, which is preferablystabilized by reduction to a secondary amine, e.g., by borohydridereduction, to form the final composite. Such site-specific linkages aredisclosed, for small molecules, in U.S. Pat. No. 4,671,958, and forlarger addends in U.S. Pat. No. 4,699,784, the Examples section of eachof which is incorporated herein by reference.

ScFvs with linkers greater than 12 amino acid residues in length (forexample, 15- or 18-residue linkers) allow interactions between the VHand VL domains on the same chain and generally form a mixture ofmonomers, dimers (termed diabodies) and small amounts of higher massmultimers, (Kortt et al., Eur J. Biochem. (1994) 221: 151-157). ScFvswith linkers of 5 or less amino acid residues, however, prohibitintramolecular pairing of the VH and VL domains on the same chain,forcing pairing with VH and VL domains on a different chain. Linkersbetween 3- and 12-residues form predominantly dimers (Atwell et al.,Protein Engineering (1999) 12: 597-604). With linkers between 0 and 2residues, trimeric (termed triabodies), tetrameric (termed tetrabodies)or higher oligomeric structures of scFvs are formed; however, the exactpatterns of oligomerization appear to depend on the composition as wellas the orientation of the V-domains, in addition to the linker length.

More recently, a novel technique for construction of mixtures ofantibodies, antibody fragments and/or other effector moieties invirtually any combination desired has been described in U.S. patentapplication Ser. No. 11/389,358 (now issued U.S. Pat. No. 7,550,143),filed Mar. 24, 2006; 11/391,584 (now issued U.S. Pat. No. 7,521,056),filed Mar. 28, 2006; 11/478,021 (now issued U.S. Pat. No. 7,534,866),filed Jun. 29, 2006; 11/633,729 (now issued U.S. Pat. No. 7,527,787),filed Dec. 5, 2006; and 11/925,408, filed Oct. 26, 2007, the Examplessection of each of which is incorporated herein by reference. Thetechnique, known generally as dock-and-lock (DNL) involves theproduction of fusion proteins that comprise at their N- or C-terminalends one of two complementary peptide sequences, called dimerization anddocking domain (DDD) and anchoring domain (AD) sequences. In preferredembodiments, the DDD sequences are derived from the regulatory subunitsof cAMP-dependent protein kinase and the AD sequence is derived from thesequence of A-kinase anchoring protein (AKAP). The DDD sequences formdimers that bind to the AD sequence, which allows formation of trimers,tetramers, hexamers or any of a variety of other complexes. By attachingeffector moieties, such as antibodies or antibody fragments, to the DDDand AD sequences, complexes may be formed of any selected combination ofantibodies or antibody fragments. The DNL complexes may be covalentlystabilized by formation of disulfide bonds or other linkages.

Bispecific antibodies comprising the antigen-binding variable regionsequences of any known anti-TAA antibody may be utilized, including butnot limited to hPAM4 (U.S. Pat. No. 7,282,567), hA20 (U.S. Pat. No.7,251,164), hA19 (U.S. Pat. No. 7,109,304), hIMMU31 (U.S. Pat. No.7,300,655), hLL1 (U.S. Pat. No. 7,312,318,), hLL2 (U.S. Pat. No.7,074,403), hMu-9 (U.S. Pat. No. 7,387,773), hL243 (U.S. patentapplication Ser. No. 11/368,296), hMN-14 (U.S. Pat. No. 6,676,924), hRS7(U.S. Pat. No. 7,238,785), hMN-3 (U.S. patent application Ser. No.10/672,278) and hR1 (U.S. Provisional Patent Application Ser. No.61/145,896, filed Jan. 20, 2009) the Examples section of each citedpatent or application incorporated herein by reference.

Other antibodies of use may be commercially obtained from a wide varietyof known sources. For example, a variety of antibody secreting hybridomalines are available from the American Type Culture Collection (ATCC,Manassas, Va.). A large number of antibodies against various diseasetargets, including but not limited to tumor-associated antigens, havebeen deposited at the ATCC and/or have published variable regionsequences and are available for use in the claimed methods andcompositions. See, e.g., U.S. Pat. Nos. 7,312,318; 7,282,567; 7,151,164;7,074,403; 7,060,802; 7,056,509; 7,049,060; 7,045,132; 7,041,803;7,041,802; 7,041,293; 7,038,018; 7,037,498; 7,012,133; 7,001,598;6,998,468; 6,994,976; 6,994,852; 6,989,241; 6,974,863; 6,965,018;6,964,854; 6,962,981; 6,962,813; 6,956,107; 6,951,924; 6,949,244;6,946,129; 6,943,020; 6,939,547; 6,921,645; 6,921,645; 6,921,533;6,919,433; 6,919,078; 6,916,475; 6,905,681; 6,899,879; 6,893,625;6,887,468; 6,887,466; 6,884,594; 6,881,405; 6,878,812; 6,875,580;6,872,568; 6,867,006; 6,864,062; 6,861,511; 6,861,227; 6,861,226;6,838,282; 6,835,549; 6,835,370; 6,824,780; 6,824,778; 6,812,206;6,793,924; 6,783,758; 6,770,450; 6,767,711; 6,764,688; 6,764,681;6,764,679; 6,743,898; 6,733,981; 6,730,307; 6,720,155; 6,716,966;6,709,653; 6,693,176; 6,692,908; 6,689,607; 6,689,362; 6,689,355;6,682,737; 6,682,736; 6,682,734; 6,673,344; 6,653,104; 6,652,852;6,635,482; 6,630,144; 6,610,833; 6,610,294; 6,605,441; 6,605,279;6,596,852; 6,592,868; 6,576,745; 6,572,856; 6,566,076; 6,562,618;6,545,130; 6,544,749; 6,534,058; 6,528,625; 6,528,269; 6,521,227;6,518,404; 6,511,665; 6,491,915; 6,488,930; 6,482,598; 6,482,408;6,479,247; 6,468,531; 6,468,529; 6,465,173; 6,461,823; 6,458,356;6,455,044; 6,455,040, 6,451,310; 6,444,206; 6,441,143; 6,432,404;6,432,402; 6,419,928; 6,413,726; 6,406,694; 6,403,770; 6,403,091;6,395,276; 6,395,274; 6,387,350; 6,383,759; 6,383,484; 6,376,654;6,372,215; 6,359,126; 6,355,481; 6,355,444; 6,355,245; 6,355,244;6,346,246; 6,344,198; 6,340,571; 6,340,459; 6,331,175; 6,306,393;6,254,868; 6,187,287; 6,183,744; 6,129,914; 6,120,767; 6,096,289;6,077,499; 5,922,302; 5,874,540; 5,814,440; 5,798,229; 5,789,554;5,776,456; 5,736,119; 5,716,595; 5,677,136; 5,587,459; 5,443,953,5,525,338, the Examples section of each of which is incorporated hereinby reference. These are exemplary only and a wide variety of otherantibodies and their hybridomas are known in the art. The skilledartisan will realize that antibody sequences or antibody-secretinghybridomas against almost any disease-associated antigen may be obtainedby a simple search of the ATCC, NCBI and/or USPTO databases forantibodies against a selected disease-associated target of interest. Theantigen binding domains of the cloned antibodies may be amplified,excised, ligated into an expression vector, transfected into an adaptedhost cell and used for protein production, using standard techniqueswell known in the art.

Pretargeting

Bispecific or multispecific antibodies may be utilized in pre-targetingtechniques. Pre-targeting is a multistep process originally developed toresolve the slow blood clearance of directly targeting antibodies, whichcontributes to undesirable toxicity to normal tissues such as bonemarrow. With pre-targeting, a radionuclide or other therapeutic agent isattached to a small delivery molecule (targetable construct ortargetable conjugate) that is cleared within minutes from the blood. Apre-targeting bispecific or multispecific antibody, which has bindingsites for the targetable construct as well as a target antigen, isadministered first, free antibody is allowed to clear from circulationand then the targetable construct is administered.

Pre-targeting methods are well known in the art, for example, asdisclosed in Goodwin et al., U.S. Pat. No. 4,863,713; Goodwin et al., J.Nucl. Med. 29:226, 1988; Hnatowich et al., J. Nucl. Med. 28:1294, 1987;Oehr et al., J. Nucl. Med. 29:728, 1988; Klibanov et al., J. Nucl. Med.29:1951, 1988; Sinitsyn et al., J. Nucl. Med. 30:66, 1989; Kalofonos etal., J. Nucl. Med. 31:1791, 1990; Schechter et al., Int. J. Cancer48:167, 1991; Paganelli et al., Cancer Res. 51:5960, 1991; Paganelli etal., Nucl. Med. Commun. 12:211, 1991; U.S. Pat. No. 5,256,395; Stickneyet al., Cancer Res. 51:6650, 1991; Yuan et al., Cancer Res. 51:3119,1991; U.S. Pat. No. 6,077,499; U.S. Ser. No. 09/597,580; U.S. Ser. No.10/361,026; U.S. Ser. No. 09/337,756; U.S. Ser. No. 09/823,746; U.S.Ser. No. 10/116,116; U.S. Ser. No. 09/382,186; U.S. Ser. No. 10/150,654;U.S. Pat. No. 6,090,381; U.S. Pat. No. 6,472,511; U.S. Ser. No.10/114,315; U.S. Provisional Application No. 60/386,411; U.S.Provisional Application No. 60/345,641; U.S. Provisional Application No.60/3328,835; U.S. Provisional Application No. 60/426,379; U.S. Ser. No.09/823,746; U.S. Ser. No. 09/337,756; U.S. Provisional Application No.60/342,103; and U.S. Pat. No. 6,962,702.

A pre-targeting method of treating or diagnosing a disease or disorderin a subject may be provided by: (1) administering to the subject abispecific antibody or antigen binding antibody fragment; (2) optionallyadministering to the subject a clearing composition, and allowing thecomposition to clear the antibody from circulation; and (3)administering to the subject the targetable construct, containing one ormore chelated or chemically bound therapeutic or diagnostic agents. Thetechnique may also be utilized for antibody dependent enzyme prodrugtherapy (ADEPT) by administering an enzyme conjugated to a targetableconstruct, followed by a prodrug that is converted into active form bythe enzyme.

Expression Vectors and Host Cells

An expression vector is a DNA molecule comprising a gene that isexpressed in a host cell. Typically, gene expression is placed under thecontrol of certain regulatory elements, including constitutive orinducible promoters, tissue-specific regulatory elements, and enhancers.Such a gene is said to be “operably linked to” the regulatory elements.A promoter is a DNA sequence that directs the transcription of astructural gene. A structural gene is a DNA sequence that is transcribedinto messenger RNA (mRNA) which is then translated into a peptide orprotein. If a promoter is an inducible promoter, then the rate oftranscription increases in response to an inducing agent. In contrast,the rate of transcription is not regulated by an inducing agent if thepromoter is a constitutive promoter. An enhancer is a DNA regulatoryelement that can increase the efficiency of transcription, regardless ofthe distance or orientation of the enhancer relative to the start siteof transcription.

An isolated DNA molecule is a fragment of DNA that is not integrated inthe chromosomal DNA of a cell or organism. In preferred embodiments, aDNA sequence to be expressed will be packaged into an expression vectorand transfected into a host cell, where it is preferably integrated intothe host cell chromosomal DNA. Methods for construction of nucleic acidsof any selected sequence, for example by chemical synthesis of shorteroligonucleotides and assembly into a protein encoding sequence, are wellknown in the art. Alternatively, DNA sequences of interest may be cutusing restriction endonucleases and spliced together to make a selectedprotein encoding sequence. Other techniques for producing specificchanges in encoded protein sequences, such as site-directed mutagenesis,are also well known.

A cloning vector is a DNA molecule, such as a plasmid, cosmid, orbacteriophage, that has the capability of replicating autonomously in ahost cell. Cloning vectors typically contain one or a small number ofrestriction endonuclease recognition sites at which foreign DNAsequences can be inserted in a determinable fashion without loss of anessential biological function of the vector, as well as a marker genethat is suitable for use in the identification and selection of cellstransformed with the cloning vector. Marker genes typically includegenes that provide resistance to tetracycline, ampicillin, kanamycin orother antibiotics. A recombinant host may be any prokaryotic oreukaryotic cell that contains either a cloning vector or expressionvector. This term also includes those prokaryotic or eukaryotic cellsthat have been genetically engineered to contain the cloned gene(s) inthe chromosome or genome of the host cell.

Suitable host cells include microbial or mammalian host cells. Apreferred host is the human cell line, PER.C6, which was developed forproduction of MAbs, and other fusion proteins. Accordingly, a preferredembodiment is a host cell comprising a DNA sequence encoding theanti-pancreatic cancer MAb, conjugate, fusion protein or fragmentsthereof. PER.C6 cells (WO 97/00326) were generated by transfection ofprimary human embryonic retina cells, using a plasmid that contained theAdserotype 5 (Ad5) E1A- and E1B-coding sequences (Ad5 nucleotides459-3510) under the control of the human phosphoglycerate kinase (PGK)promoter.

Other mammalian host cells may be used, including myeloma cells, such asSP2/0 cells, and NS0 cells, as well as Chinese Hamster Ovary (CHO)cells, hybridoma cell lines and other mammalian host cell useful forexpressing antibodies. Also particularly useful to express mAbs andother fusion proteins are Sp2/0 cells transfected with an apoptosisinhibitor, such as a Bcl-EEE gene, and adapted to grow and be furthertransfected in serum free conditions, as described in U.S. patentapplication Ser. Nos. 11/187,863 (now issued U.S. Pat. No. 7,531,327),filed Jul. 25, 2005; 11/487,215 (now issued U.S. Pat. No. 7,537,930),filed Jul. 14, 2006; and 11/877,728, filed Oct. 24, 2007, the Examplessection of each of which is incorporated herein by reference.) Incertain cases, the apoptosis inhibitor and/or structural gene to beexpressed may be amplified by exposing the host cell to an appropriateconcentration of methotrexate. Sp2/0 cells transfected with Bcl-EEE andconditioned to grow in serum-free medium have been reported to exhibitedprolonged longevity in culture, higher cell density, and significantlyhigher rates of protein production (U.S. Pat. No. 7,531,327; U.S. Pat.No. 7,537,930); U.S. Ser. No. 11/877,728, filed Oct. 24, 2007).

Antibody Use for Treatment and Diagnosis

Certain embodiments concern methods of diagnosing or treating amalignancy in a subject, comprising administering to the subject ananti-pancreatic cancer MAb, fusion protein or fragment thereof, whereinthe MAb, fusion protein or fragment is bound to at least one diagnosticand/or therapeutic agent. Also preferred is a method for diagnosing ortreating cancer, comprising administering to a subject a multivalent,multispecific antibody or fragment thereof comprising one or moreantigen binding sites toward a PAM4 antigen and one or more haptenbinding sites, waiting a sufficient amount of time for non-boundantibody to clear the subject's blood stream; and then administering tothe subject a carrier molecule comprising a diagnostic agent, atherapeutic agent, or a combination thereof, that binds to thehapten-binding site of the localized antibody. In a more preferredembodiment, the cancer is a non-endocrine pancreatic cancer.

The use of MAbs for in vitro diagnosis is well-known. See, for example,Carlsson et al., Bio/Technology 7 (6): 567 (1989). For example, MAbs canbe used to detect the presence of a tumor-associated antigen in tissuefrom biopsy samples. MAbs also can be used to measure the amount oftumor-associated antigen in clinical fluid samples using techniques suchas radioimmunoassay, enzyme-linked immunosorbent assay, and fluorescenceimmunoassay.

Conjugates of tumor-targeted MAbs and toxins can be used to selectivelykill cancer cells in vivo (Spalding, Bio/Technology 9(8): 701 (1991);Goldenberg, Scientific American Science & Medicine 1(1): 64 (1994)). Forexample, therapeutic studies in experimental animal models havedemonstrated the anti-tumor activity of antibodies carrying cytotoxicradionuclides. (Goldenberg et al., Cancer Res. 41: 4354 (1981), Cheunget al., J. Nat'l Cancer Inst. 77: 739 (1986), and Senekowitsch et al.,J. Nucl. Med. 30: 531 (1989)). In a preferred embodiment, the conjugatecomprises a ⁹⁰Y-labeled hPAM4 antibody. The conjugate may optionally beadministered in conjunction with one or more other therapeutic agents.In a preferred embodiment, ⁹⁰Y-labeled hPAM4 is administered togetherwith gemcitabine or 5-fluorouracil to a patient with pancreatic cancer.In a further preferred embodiment, ⁹⁰Y is conjugated to a DOTA chelatefor attachment to hPAM4. In a still further preferred embodiment, the⁹⁰Y-DOTA-hPAM4 is combined with gemcitabine in fractionated dosescomprising a treatment cycle, such as with repeated, lower, less-toxicdoses of gemcitabine combined with lower, fractionated doses of⁹⁰Y-DOTA-hPAM4.

As tolerated, repeated cycles of this fractionated dose schedule areindicated. By way of example, 4 weekly doses of 200 mg/m² of gemcitabineare combined with three weekly doses of 8 mg/m² of ⁹⁰Y-DOTA-hPAM4, withthe latter commencing in the second week of gemcitabine administration;this constitutes a single therapy cycle. Still other doses, higher andlower of each component, may constitute a fractionated dose, which isdetermined by conventional means of assessing hematopoietic toxicity,since myelosuppressive effects of both agents can be cumulative. Askilled physician in such therapy interventions can adjust these dosesbased on the patient's bone marrow status and general health statusbased on many factors, including prior exposure to myelosuppressivetherapeutic agents. These principles can also apply to combinations ofradiolabeled hPAM4 with other therapeutic agents, includingradiosensitizing drugs such as 5-fluorouracil and cisplatin.

Chimeric, humanized and human antibodies and fragments thereof have beenused for in vivo therapeutic and diagnostic methods. Accordinglycontemplated is a method of delivering a diagnostic or therapeuticagent, or a combination thereof, to a target comprising (i) providing acomposition that comprises an anti-pancreatic cancer antibody orfragment thereof, such as a chimeric, humanized or human PAM4 antibody,conjugated to at least one diagnostic and/or therapeutic agent and (ii)administering to a subject the diagnostic or therapeutic antibodyconjugate. In a preferred embodiment, the anti-pancreatic cancerantibodies and fragments thereof are humanized or fully human.

Also described herein is a cancer cell targeting diagnostic ortherapeutic conjugate comprising, for example, a chimeric, humanized orhuman PAM4 MAb or fragment thereof bound to at least one diagnostic orat least one therapeutic agent. Preferably, the diagnostic conjugate isa photoactive diagnostic agent, an ultrasound detectable agent, an MRIcontrast agent or a PET radionuclide, such as ¹⁸F or ⁶⁸Ga. Stillpreferred, the diagnostic agent is a radionuclide with an energy between20 and 4,000 keV.

Another embodiment concerns a method for treating a malignancycomprising administering a naked anti-pancreatic cancer antibody,antibody fragment or fusion protein, such as a PAM4 antibody, eitheralone or in conjunction with one or more other therapeutic agents. Theother therapeutic agent may be added before, simultaneously with orafter the antibody. In a preferred embodiment, the therapeutic agent isgemcitabine, and in a more preferred embodiment, gemcitabine is givenwith the hPAM4 radioconjugate in a fractionated dose schedule at lowerdoses than the conventional 800-1,000 mg/m² doses of gemcitabine givenweekly for 6 weeks. For example, when combined with fractionatedtherapeutic doses of ⁹⁰Y-PAM4, repeated fractionated doses intended tofunction as a radiosensitizing agent of 200-380 mg/m² gemcitabine areinfused. The skilled artisan will realize that the antibodies, fusionproteins and/or fragments thereof described and claimed herein may beadministered with any known or described therapeutic agent, includingbut not limited to heat shock protein 90 (Hsp90)

The compositions for treatment contain at least one humanized or fullyhuman anti-pancreatic cancer antibody or fragment thereof either aloneand unconjugated, or conjugated or unconjugated and in combination withother antibodies or fragments thereof, such as other humanized orchimeric antibodies, human antibodies, therapeutic agents orimmunomodulators. Naked or conjugated antibodies to the same ordifferent epitope or antigen may also be combined with one or more ofthe anti-pancreatic cancer antibodies or fragments thereof.

Accordingly, the present invention contemplates the administration ofanti-pancreatic cancer antibodies and fragments thereof, includingfusion proteins and fragments thereof, alone, as a naked antibody orantibody fragment, or administered as a multimodal therapy. Preferably,the antibody is a humanized or fully human PAM4 antibody or fragmentthereof. Multimodal therapies further include immunotherapy with a nakedanti-pancreatic cancer antibody supplemented with administration ofother antibodies in the form of naked antibodies, fusion proteins, or asimmunoconjugates. For example, a humanized or fully human PAM4 antibodymay be combined with another naked antibody, or a humanized PAM4 orother antibody conjugated to an isotope, one or more chemotherapeuticagents, cytokines, toxins or a combination thereof. For example, thepresent invention contemplates treatment of a naked or conjugated PAM4antibody or fragments thereof before, in combination with, or afterother pancreatic tumor associated antibodies such as CA19.9, DUPAN2,SPAN1, Nd2, B72.3, CC49, anti-Le^(a) antibodies, and antibodies to otherLewis antigens (e.g., Le(y)), as well as antibodies againstcarcinoembryonic antigen (CEA or CEACAM5), CEACAM6, colon-specificantigen-p (CSAp), MUC-1, MUC-2, MUC-3, MUC-4, MUC-5ac, MUC-16, MUC-17,HLA-DR, CD40, CD74, CD138, HER2/neu, EGFR, EGP-1, EGP-2, angiogenesisfactors (e.g., VEGF, P1GF), insulin-like growth factor (ILGF), tenascin,platelet-derived growth factor, and IL-6, as well as products ofoncogenes (e.g., bcl-2, Kras, p53), cMET, and antibodies against tumornecrosis substances. These solid tumor antibodies may be naked orconjugated to, inter glia, drugs, toxins, isotopes, radionuclides orimmunomodulators. Many different antibody combinations may beconstructed and used in either naked or conjugated form. Alternatively,different naked antibody combinations may be employed for administrationin combination with other therapeutic agents, such as a cytotoxic drugor with radiation, given consecutively, simultaneously, or sequentially.

The antibodies described herein directly target PAM4 positive tumors.The antibodies bind selectively to pancreatic cancer or other cancerantigens and as the number of binding sites on the molecule increases,the affinity for the target cell increases and a longer residence timeis observed at the desired location. Moreover, non-antigen boundmolecules are cleared from the body quickly and exposure of normaltissues is minimized. A use of multispecific antibodies is in AESsystems, where anti-PAM4 antibodies pre-target positive tumors forsubsequent specific delivery of diagnostic or therapeutic agents. Theagents may be carried by histamine succinyl glycyl (HSG) containingpeptides. The murine monoclonal antibody designated 679 binds with highaffinity to molecules containing the tri-peptide HSG hapten (Morel etal, Molecular Immunology, 27, 995-1000, 1990) and may be used to form abispecific antibody with hPAM4; but even more preferred is the use of ahumanized version of 679. Alternative haptens may also be utilized, suchas In-DTPA or NOTA. The bispecific antibodies bind selectively totargeted antigens allowing for increased affinity and a longer residencetime at the desired location. Moreover, non-antigen bound antibodies arecleared from the body quickly and exposure of normal tissues isminimized. PAM4 and other antibodies against pancreatic cancer can beused to diagnose and/or treat mammalian cancer. Diagnosis requires thefurther step of detecting the bound, labeled antibodies or fragmentsusing known techniques.

In the context of this application, the terms “diagnosis” or “detection”can be used interchangeably. Whereas diagnosis usually refers todefining a tissue's specific histological status, detection recognizesand locates a tissue, lesion or organism containing a particularantigen.

Administration of the antibodies and their fragments can be effected byintravenous, intraarterial, intraperitoneal, intramuscular,subcutaneous, intrapleural, intrathecal, perfusion through a regionalcatheter, or direct intralesional injection. When administering theantibody by injection, the administration may be by continuous infusionor by single or multiple boluses.

Naked Antibody Therapy

The efficacy of the naked anti-pancreatic cancer antibodies and theirfragments can be enhanced by supplementing these naked antibodies withone or more other naked antibodies, or with one or more immunoconjugatesconjugated with one or more therapeutic agents, including drugs, toxins,immunomodulators, hormones, oligonucleotides, hormone antagonists,enzymes, enzyme inhibitors, therapeutic radionuclides, angiogenesisinhibitors, etc., administered concurrently or sequentially or accordingto a prescribed dosing regimen. Alternatively, naked antibodies may beadministered in conjunction with therapeutic agents that are notattached to other antibodies. Antibodies that may be used to supplementthe naked anti-pancreatic cancer antibodies may be directed againsteither the same cancer cells or against immunomodulator cells (e.g.,CD40+ cells) that can be recruited to enhance the antitumor effects ofthe naked antibodies of choice.

Immunoconjugates, Therapeutic and Diagnostic Agents

Anti-pancreatic cancer antibodies and fragments thereof may beconjugated to at least one therapeutic and/or diagnostic agent fortherapy or diagnosis. For immunotherapy, the objective is to delivercytotoxic doses of radioactivity, toxin, antibody and/or drug to targetcells, while minimizing exposure to non-target tissues. Preferably,anti-pancreatic cancer antibodies are be used to diagnose and/or treatpancreatic tumors.

Any of the antibodies, antibody fragments and fusion proteins can beconjugated with one or more therapeutic or diagnostic agents, using avariety of techniques known in the art. One or more therapeutic ordiagnostic agents may be attached to each antibody, antibody fragment orfusion protein. If the Fc region is absent (for example with certainantibody fragments), it is possible to introduce a carbohydrate moietyinto the light chain variable region of either an antibody or antibodyfragment to which a therapeutic or diagnostic agent may be attached.See, for example, Leung et al., J Immunol. 154: 5919 (1995); Hansen etal., U.S. Pat. No. 5,443,953 (1995), Leung et al., U.S. Pat. No.6,254,868, the Examples section of each patent incorporated herein byreference.

Methods for conjugating peptides to antibody components via an antibodycarbohydrate moiety are well-known to those of skill in the art. See,for example, Shih et al., Int J Cancer 41: 832 (1988); Shih et al., IntJ Cancer 46: 1101 (1990); and Shih et al., U.S. Pat. No. 5,057,313, theExamples section of which is incorporated herein by reference. Thegeneral method involves reacting an antibody component having anoxidized carbohydrate portion with a carrier polymer that has at leastone free amine function and that is loaded with a plurality oftherapeutic agents, such as peptides or drugs. This reaction results inan initial Schiff base (imine) linkage, which can be stabilized byreduction to a secondary amine to form the final conjugate.

Antibody fusion proteins or multispecific antibodies comprise two ormore antibodies or fragments thereof, each of which may be attached toat least one therapeutic agent and/or diagnostic agent. Accordingly, oneor more of the antibodies or fragments thereof of the antibody fusionprotein can have more than one therapeutic and/or diagnostic agentattached. Further, the therapeutic agents do not need to be the same butcan be different therapeutic agents, for example, one can attach a drugand a radioisotope to the same fusion protein. Particularly, an IgG canbe radiolabeled with ¹³¹I and attached to a drug. The ¹³¹I can beincorporated into the tyrosine of the IgG and the drug attached to theepsilon amino group of the IgG lysines. Both therapeutic and diagnosticagents also can be attached to reduced SH groups and to the carbohydrateside chains of antibodies. Alternatively, a bispecific antibody maycomprise one antibody or fragment thereof against a disease antigen andanother against a hapten attached to a targetable construct, for use inpretargeting techniques as discussed above.

A wide variety of diagnostic and therapeutic reagents can beadministered concurrently or sequentially, or advantageously conjugatedto the antibodies of the invention, for example, drugs, toxins,oligonucleotides, immunomodulators, hormones, hormone antagonists,enzymes, enzyme inhibitors, radionuclides, angiogenesis inhibitors, etc.The therapeutic agents recited here are those agents that also areuseful for administration separately with a naked antibody as describedabove. Therapeutic agents include, for example, chemotherapeutic drugssuch as vinca alkaloids, anthracyclines, gemcitabine, epidophyllotoxins,taxanes, antimetabolites, alkylating agents, antibiotics, SN-38, COX-2inhibitors, antimitotics, antiangiogenic and apoptotoic agents,particularly doxorubicin, methotrexate, taxol, CPT-11, camptothecans,proteosome inhibitors, mTOR inhibitors, HDAC inhibitors, tyrosine kinaseinhibitors, and others from these and other classes of anticanceragents, and the like. Other useful cancer chemotherapeutic drugs foradministering concurrently or sequentially, or for the preparation ofimmuno conjugates and antibody fusion proteins include nitrogenmustards, alkyl sulfonates, nitrosoureas, triazenes, folic acid analogs,COX-2 inhibitors, antimetabolites, pyrimidine analogs, purine analogs,platinum coordination complexes, mTOR inhibitors, tyrosine kinaseinhibitors, proteosome inhibitors, HDAC inhibitors, camptothecins,hormones, and the like. Suitable chemotherapeutic agents are describedin REMINGTON'S PHARMACEUTICAL SCIENCES, 19th Ed. (Mack Publishing Co.1995), and in GOODMAN AND GILMAN'S THE PHARMACOLOGICAL BASIS OFTHERAPEUTICS, 7th Ed. (MacMillan Publishing Co. 1985), as well asrevised editions of these publications. Other suitable chemotherapeuticagents, such as experimental drugs, are known to those of skill in theart.

In a preferred embodiment, conjugates of camptothecins and relatedcompounds, such as SN-38, may be conjugated to hPAM4 or otheranti-pancreatic cancer antibodies, for example as disclosed in U.S.patent application Ser. No. 12/026,811, filed Feb. 6, 2008; and11/388,032, filed Mar. 23, 2006, the Examples section of each of whichis incorporated herein by reference.

In another preferred embodiment, an hPAM4 antibody is conjugated togemcitabine. In another embodiment, gemcitabine is given before, after,or concurrently with a naked or conjugated chimeric, humanized or humanPAM4 antibody. Preferably, the conjugated hPAM4 antibody or antibodyfragment is conjugated to a radionuclide.

A toxin can be of animal, plant or microbial origin. A toxin, such asPseudomonas exotoxin, may also be complexed to or form the therapeuticagent portion of an immunoconjugate of the anti-pancreatic cancer andhPAM4 antibodies. Other toxins suitably employed in the preparation ofsuch conjugates or other fusion proteins, include ricin, abrin,ribonuclease (RNase), DNase I, Staphylococcal enterotoxin-A, pokeweedantiviral protein, gelonin, diphtheria toxin, Pseudomonas exotoxin, andPseudomonas endotoxin. See, for example, Pastan et al., Cell 47:641(1986), Goldenberg, CA—A Cancer Journal for Clinicians 44:43 (1994),Sharkey and Goldenberg, CA—A Cancer Journal for Clinicians 56:226(2006). Additional toxins suitable for use are known to those of skillin the art and are disclosed in U.S. Pat. No. 6,077,499, the Examplessection of which is incorporated herein by reference.

An immunomodulator, such as a cytokine, may also be conjugated to, orform the therapeutic agent portion of the immunoconjugate, or may beadministered with, but unconjugated to, an antibody, antibody fragmentor fusion protein. The fusion protein may comprise one or moreantibodies or fragments thereof binding to different antigens. Forexample, the fusion protein may bind the PAM4 antigen as well asimmunomodulating cells or factors. Alternatively, subjects can receive anaked antibody, antibody fragment or fusion protein and a separatelyadministered cytokine, which can be administered before, concurrently orafter administration of the naked antibodies. As used herein, the term“immunomodulator” includes a cytokine, a lymphokine, a monokine, a stemcell growth factor, a lymphotoxin, a hematopoietic factor, a colonystimulating factor (CSF), an interferon (IFN), parathyroid hormone,thyroxine, insulin, proinsulin, relaxin, prorelaxin, folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH),luteinizing hormone (LH), hepatic growth factor, prostaglandin,fibroblast growth factor, prolactin, placental lactogen, OB protein, atransforming growth factor (TGF), TGF-α, TGF-β, insulin-like growthfactor (ILGF), erythropoietin, thrombopoietin, tumor necrosis factor(TNF), TNF-α, TNF-β, a mullerian-inhibiting substance, mousegonadotropin-associated peptide, inhibin, activin, vascular endothelialgrowth factor, integrin, interleukin (IL), granulocyte-colonystimulating factor (G-CSF), granulocyte macrophage-colony stimulatingfactor (GM-CSF), interferon-α, interferon-β, interferon-γ, S1 factor,IL-1, IL-1cc, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10,IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18 IL-21 and IL-25,LIF, kit-ligand, FLT-3, angiostatin, thrombospondin, endostatin and LT,and the like.

Alternatively, the antibodies and fragments can be detectably labeled bylinking the antibody to an enzyme. When the antibody-enzyme conjugate isincubated in the presence of the appropriate substrate, the enzymemoiety reacts with the substrate to produce a chemical moiety which canbe detected, for example, by spectrophotometric, fluorometric or visualmeans. Examples of enzymes that can be used to detectably label antibodyinclude malate dehydrogenase, staphylococcal nuclease, delta-V-steroidisomerase, yeast alcohol dehydrogenase, alpha-glycerophosphatedehydrogenase, triose phosphate isomerase, horseradish peroxidase,alkaline phosphatase, asparaginase, glucose oxidase,alpha-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphatedehydrogenase, glucoamylase and acetylcholinesterase.

A therapeutic or diagnostic agent can be attached at the hinge region ofa reduced antibody component via disulfide bond formation. As analternative, such agents can be attached to the antibody component usinga heterobifunctional cross-linker, such as N-succinyl342-pyridyldithio)proprionate (SPDP). Yu et al., Int. J. Cancer 56: 244(1994). General techniques for such conjugation are well-known in theart. See, for example, Wong, CHEMISTRY OF PROTEIN CONJUGATION ANDCROSS-LINKING (CRC Press 1991); Upeslacis et al., “Modification ofAntibodies by Chemical Methods,” in MONOCLONAL ANTIBODIES: PRINCIPLESAND APPLICATIONS, Birch et al. (eds.), pages 187-230 (Wiley-Liss, Inc.1995); Price, “Production and Characterization of SyntheticPeptide-Derived Antibodies,” in MONOCLONAL ANTIBODIES: PRODUCTION,ENGINEERING AND CLINICAL APPLICATION, Ritter et al. (eds.), pages 60-84(Cambridge University Press 1995). Alternatively, the therapeutic ordiagnostic agent can be conjugated via a carbohydrate moiety in the Fcregion of the antibody. The carbohydrate group can be used to increasethe loading of the same agent that is bound to a thiol group, or thecarbohydrate moiety can be used to bind a different peptide.

The immunoconjugate may comprise one or more radioactive isotopes usefulfor detecting diseased tissue. Particularly useful diagnosticradionuclides include, but are not limited to, ¹¹⁰In, ¹¹¹In, ¹⁷⁷Lu, ¹⁸F,⁵²Fe, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁸⁶Y, ⁹⁰Y, ⁸⁹Zr, ^(94m)Tc, ⁹⁴Tc,^(99m)Tc, ¹²⁰I, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹⁵⁴⁻¹⁵⁸Gd, ³²P, ¹¹C, ¹³N, ¹⁵O,¹⁸⁶Re, ¹⁸⁸Re, ⁵¹Mn, ^(52m)Mn, ⁵⁵Co, ⁷²As, ⁷⁵Br, ⁷⁶Br, ^(82m)Rb, ⁸³Sr, orother gamma-, beta-, or positron-emitters, preferably with a decayenergy in the range of 20 to 4,000 keV, more preferably in the range of25 to 4,000 keV, and even more preferably in the range of 25 to 1,000keV, and still more preferably in the range of 70 to 700 keV. Totaldecay energies of useful positron-emitting radionuclides are preferably<2,000 keV, more preferably under 1,000 keV, and most preferably <700keV. Radionuclides useful as diagnostic agents utilizing gamma-raydetection include, but are not limited to: ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁶⁷Cu,⁶⁷Ga, ⁷⁵Se, ⁹⁷Ru, ^(99m)Tc, ¹¹¹In, ^(114m)In, ¹²³I, ¹²⁵I, ¹³¹I, ¹⁶⁹Yb,¹⁹⁷Hg, and ²⁰¹Tl. Decay energies of useful gamma-ray emittingradionuclides are preferably 20-2000 keV, more preferably 60-600 keV,and most preferably 100-300 keV.

The immunoconjugate may comprise one or more radioactive isotopes usefulfor treating diseased tissue. Particularly useful therapeuticradionuclides include, but are not limited to ¹¹¹In, ¹⁷⁷Lu, ²¹²Bi,²¹³Bi, ²¹¹At, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁹⁰Y, ¹²⁵I, ¹³¹I, ³²P, ³³P, ⁴⁷Sc, ¹¹¹Ag,⁶⁷Ga, ¹⁴²Pr, ¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re, ²¹²Pb,²²³Ra, ²²⁵Ac, ⁵⁹Fe, ⁷⁵Se, ⁷⁷As, ⁸⁹Sr, ⁹⁹Mo, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹⁴³Pr, ¹⁴⁹Pm,¹⁶⁹Er, ¹⁹⁴Ir, ¹⁹⁸Au, ¹⁹⁹Au, and ²¹¹Pb. The therapeutic radionuclidepreferably has a decay energy in the range of 20 to 6,000 keV,preferably in the ranges 60 to 200 keV for an Auger emitter, 100-2,500keV for a beta emitter, and 4,000-6,000 keV for an alpha emitter.Maximum decay energies of useful beta-particle-emitting nuclides arepreferably 20-5,000 keV, more preferably 100-4,000 keV, and mostpreferably 500-2,500 keV. Also preferred are radionuclides thatsubstantially decay with Auger-emitting particles. For example, Co-58,Ga-67, Br-80m, Tc-99m, Rh-103m, Pt-109, In-111, Sb-119, I-125, Ho-161,Os-189m and Ir-192. Decay energies of useful beta-particle-emittingnuclides are preferably <1,000 keV, more preferably <100 keV, and mostpreferably <70 keV. Also preferred are radionuclides that substantiallydecay with generation of alpha-particles. Such radionuclides include,but are not limited to: Dy-152, At-211, Bi-212, Ra-223, Rn-219, Po-215,Bi-211, Ac-225, Fr-221, At-217, Bi-213 and Fm-255. Decay energies ofuseful alpha-particle-emitting radionuclides are preferably 2,000-10,000keV, more preferably 3,000-8,000 keV, and most preferably 4,000-7,000keV.

For example, ⁶⁷Cu, considered one of the more promising radioisotopesfor radioimmunotherapy due to its 61.5-hour half-life and abundantsupply of beta particles and gamma rays, can be conjugated to anantibody using the chelating agent,p-bromoacetamido-benzyl-tetraethylaminetetraacetic acid (TETA). Chase,supra. Alternatively, ⁹⁰Y, which emits an energetic beta particle, canbe coupled to an antibody, antibody fragment or fusion protein, usingdiethylenetriaminepentaacetic acid (DTPA), or more preferably usingDOTA. Methods of conjugating ⁹⁰Y to antibodies or targetable constructsare known in the art and any such known methods may be used. (See, e.g.,U.S. Pat. No. 7,259,249, the Examples section of which is incorporatedherein by reference. See also Linden et al., Clin Cancer Res.11:5215-22, 2005; Sharkey et al., J Nucl Med. 46:620-33, 2005; Sharkeyet al., J Nucl Med. 44:2000-18, 2003.)

Additional potential therapeutic radioisotopes include ¹¹C, ¹³N, ¹⁵O,⁷⁵Br, ¹⁹⁸Au, ²²⁴Ac, ¹²⁶I, ¹³³I, ⁷⁷Br, ^(113m)In, ⁹⁵Ru, ⁹⁷Ru, ¹⁰³Ru,¹⁰⁵Ru, ¹⁰⁷Hg, ²⁰³Hg, ^(121m)Te, ^(122m)Te, ^(125m)Te, ¹⁶⁵Tm, ¹⁶⁷Tm,¹⁶⁸Tm, ¹⁹⁷Pt, ¹⁰⁹Pd, ¹⁰⁵Rh, ¹⁴²Pr, ¹⁴³Pr, ¹⁶¹Tb, ¹⁶⁶Ho, ¹⁹⁹Au, ⁵⁷Co,⁵⁸Co, ⁵¹Cr, ⁵⁹Fe, ⁷⁵Se, ²⁰¹Tl, ²²⁵Ac, ⁷⁶Br, ¹⁶⁹Yb, and the like.

In another embodiment, a radiosensitizer can be used in combination witha naked or conjugated antibody or antibody fragment. For example, theradiosensitizer can be used in combination with a radiolabeled antibodyor antibody fragment. The addition of the radiosensitizer can result inenhanced efficacy when compared to treatment with the radiolabeledantibody or antibody fragment alone. Radiosensitizers are described inD. M. Goldenberg (ed.), CANCER THERAPY WITH RADIOLABELED ANTIBODIES, CRCPress (1995). Other typical radionsensitizers of interest for use withthis technology include gemcitabine, 5-fluorouracil, and cisplatin, andhave been used in combination with external irradiation in the therapyof diverse cancers, including pancreatic cancer. Therefore, we havestudied the combination of gemcitabine at what is believed to beradiosensitizing doses (once weekly 200 mg/m² over 4 weeks) ofgemcitabine combined with fractionated doses of ⁹⁰Y-hPAM4, and haveobserved objective evidence of pancreatic cancer reduction after asingle cycle of this combination that proved to be well-tolerated (nograde 3-4 toxicities by NCI-CTC v. 3 standard).

Antibodies or fragments thereof that have a boron addend-loaded carrierfor thermal neutron activation therapy will normally be effected insimilar ways. However, it will be advantageous to wait untilnon-targeted immunoconjugate clears before neutron irradiation isperformed. Clearance can be accelerated using an anti-idiotypic antibodythat binds to the anti-pancreatic cancer antibody. See U.S. Pat. No.4,624,846 for a description of this general principle. For example,boron addends such as carboranes, can be attached to antibodies.Carboranes can be prepared with carboxyl functions on pendant sidechains, as is well-known in the art. Attachment of carboranes to acarrier, such as aminodextran, can be achieved by activation of thecarboxyl groups of the carboranes and condensation with amines on thecarrier. The intermediate conjugate is then conjugated to the antibody.After administration of the antibody conjugate, a boron addend isactivated by thermal neutron irradiation and converted to radioactiveatoms which decay by alpha-emission to produce highly toxic, short-rangeeffects.

Methods of diagnosing cancer in a subject may be accomplished byadministering a diagnostic immunoconjugate and detecting the diagnosticlabel attached to an immunoconjugate that is localized to a cancer ortumor. The antibodies, antibody fragments and fusion proteins may beconjugated to the diagnostic agent or may be administered in apretargeting technique using targetable constructs attached to adiagnostic agent. Radioactive agents that can be used as diagnosticagents are discussed above. A suitable non-radioactive diagnostic agentis a contrast agent suitable for magnetic resonance imaging, X-rays,computed tomography or ultrasound. Magnetic imaging agents include, forexample, non-radioactive metals, such as manganese, iron and gadolinium,complexed with metal-chelate combinations that include 2-benzyl-DTPA andits monomethyl and cyclohexyl analogs. See U.S. Ser. No. 09/921,290filed on Oct. 10, 2001, the Examples section of which is incorporatedherein by reference. Other imaging agents such as PET scanningnucleotides, preferably ¹⁸F, may also be used.

Contrast agents, such as MRI contrast agents, including, for example,gadolinium ions, lanthanum ions, dysprosium ions, iron ions, manganeseions or other comparable labels, CT contrast agents, and ultrasoundcontrast agents may be used as diagnostic agents. Paramagnetic ionssuitable for use include chromium (III), manganese (II), iron (III),iron (II), cobalt (II), nickel (II), copper (II), neodymium (III),samarium (III), ytterbium (III), gadolinium (III), vanadium (II),terbium (III), dysprosium (III), holmium (III) and erbium (III), withgadolinium being particularly preferred.

Ions useful in other contexts, such as X-ray imaging, include but arenot limited to lanthanum (III), gold (III), lead (II) and bismuth (III).Fluorescent labels include rhodamine, fluorescein and renographin.Rhodamine and fluorescein are often linked via an isothiocyanateintermediate.

Metals are also useful in diagnostic agents, including those formagnetic resonance imaging techniques. These metals include, but are notlimited to: gadolinium, manganese, iron, chromium, copper, cobalt,nickel, dysprosium, rhenium, europium, terbium, holmium and neodymium.In order to load an antibody with radioactive metals or paramagneticions, it may be necessary to react it with a reagent having a long tailto which are attached a multiplicity of chelating groups for binding theions. Such a tail can be a polymer such as a polylysine, polysaccharide,or other derivatized or derivatizable chain having pendant groups towhich can be bound chelating groups such as, e.g.,ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaaceticacid (DTPA), porphyrins, polyamines, crown ethers,bis-thiosemicarbazones, polyoximes, and like groups known to be usefulfor this purpose.

Chelates are coupled to an antibody, fusion protein, or fragmentsthereof using standard chemistries. The chelate is normally linked tothe antibody by a group which enables formation of a bond to themolecule with minimal loss of immunoreactivity and minimal aggregationand/or internal cross-linking. Other, more unusual, methods and reagentsfor conjugating chelates to antibodies are disclosed in U.S. Pat. No.4,824,659 to Hawthorne, entitled “Antibody Conjugates”, issued Apr. 25,1989, the Examples section of which is incorporated herein by reference.Particularly useful metal-chelate combinations include 2-benzyl-DTPA andits monomethyl and cyclohexyl analogs, used with diagnostic isotopes inthe general energy range of 20 to 2,000 keV. The same chelates, whencomplexed with non-radioactive metals, such as manganese, iron andgadolinium are useful for MRI. Macrocyclic chelates such as NOTA, DOTA,and TETA are of use with a variety of metals and radiometals, mostparticularly with radionuclides of gallium, yttrium and copper,respectively. Such metal-chelate complexes can be made very stable bytailoring the ring size to the metal of interest. Other ring-typechelates such as macrocyclic polyethers, which are of interest forstably binding nuclides, such as 223Ra for RAIT are encompassed by theinvention.

Radiopaque and contrast materials are used for enhancing X-rays andcomputed tomography, and include iodine compounds, barium compounds,gallium compounds, thallium compounds, etc. Specific compounds includebarium, diatrizoate, ethiodized oil, gallium citrate, iocarmic acid,iocetamic acid, iodamide, iodipamide, iodoxamic acid, iogulamide,iohexyl, iopamidol, iopanoic acid, ioprocemic acid, iosefamic acid,ioseric acid, iosulamide meglumine, iosemetic acid, iotasul, iotetricacid, iothalamic acid, iotroxic acid, ioxaglic acid, ioxotrizoic acid,ipodate, meglumine, metrizamide, metrizoate, propyliodone, and thallouschloride.

The antibodies, antibody fragments and fusion proteins also can belabeled with a fluorescent compound. The presence of afluorescent-labeled MAb is determined by exposing the antibody to lightof the proper wavelength and detecting the resultant fluorescence.Fluorescent labeling compounds include Alexa 350, Alexa 430, AMCA,aminoacridine, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G,BODIPY-TMR, BODIPY-TRX, 5-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein, 5-carboxy-2′,4′,5′,7′-tetrachlorofluorescein,5-carboxyfluorescein, 5-carboxyrhodamine, 6-carboxyrhodamine,6-carboxytetramethyl amino, Cascade Blue, Cy2, Cy3, Cy5,6-FAM, dansylchloride, Fluorescein, fluorescein isothiocyanate, fluorescamine, HEX,6-JOE, NBD (7-nitrobenz-2-oxa-1,3-diazole), Oregon Green 488, OregonGreen 500, Oregon Green 514, Pacific Blue, phthalic acid, terephthalicacid, isophthalic acid, cresyl fast violet, cresyl blue violet,brilliant cresyl blue, para-aminobenzoic acid, erythrosine,phthalocyanines, phthaldehyde, azomethines, cyanines, xanthines,succinylfluoresceins, rare earth metal cryptates, europiumtrisbipyridine diamine, a europium cryptate or chelate, diamine,dicyanins, La Jolla blue dye, allopycocyanin, allococyanin B,phycocyanin C, phycocyanin R, thiamine, phycoerythrocyanin,phycoerythrin R, REG, Rhodamine Green, rhodamine isothiocyanate,Rhodamine Red, ROX, TAMRA, TET, TRIT (tetramethyl rhodamine isothiol),Tetramethylrhodamine, and Texas Red. Fluorescently-labeled antibodiesare particularly useful for flow cytometry analysis, but can also beused in endoscopic and intravascular detection methods.

Alternatively, the antibodies, antibody fragments and fusion proteinscan be detectably labeled by coupling the antibody to a chemiluminescentcompound. The presence of the chemiluminescent-tagged MAb is determinedby detecting the presence of luminescence that arises during the courseof a chemical reaction. Examples of chemiluminescent labeling compoundsinclude luminol, isoluminol, an aromatic acridinium ester, an imidazole,an acridinium salt and an oxalate ester.

Similarly, a bioluminescent compound can be used to label the antibodiesand fragments there. Bioluminescence is a type of chemiluminescencefound in biological systems in which a catalytic protein increases theefficiency of the chemiluminescent reaction. The presence of abioluminescent protein is determined by detecting the presence ofluminescence. Bioluminescent compounds that are useful for labelinginclude luciferin, luciferase and aequorin.

Accordingly, a method of diagnosing a malignancy in a subject isdescribed, comprising performing an in vitro diagnosis assay on aspecimen (fluid, tissue or cells) from the subject with a compositioncomprising an anti-pancreatic cancer MAb, fusion protein or fragmentthereof. Immunohistochemistry can be used to detect the presence of PAM4antigen in a cell or tissue by the presence of bound antibody.Preferably, the malignancy that is being diagnosed is a cancer. Mostpreferably, the cancer is pancreatic cancer.

Additionally, a chelator such as DTPA, DOTA, TETA, or NOTA or a suitablepeptide, to which a detectable label, such as a fluorescent molecule, orcytotoxic agent, such as a heavy metal or radionuclide, can beconjugated to a subject antibody. For example, a therapeutically usefulimmuno conjugate can be obtained by conjugating a photoactive agent ordye to an antibody fusion protein. Fluorescent compositions, such asfluorochrome, and other chromogens, or dyes, such as porphyrinssensitive to visible light, have been used to detect and to treatlesions by directing the suitable light to the lesion. In therapy, thishas been termed photoradiation, phototherapy, or photodynamic therapy(Jori et al. (eds.), PHOTODYNAMIC THERAPY OF TUMORS AND OTHER DISEASES(Libreria Progetto 1985); van den Bergh, Chem. Britain 22:430 (1986)).Moreover, monoclonal antibodies have been coupled with photoactivateddyes for achieving phototherapy. Mew et al., J. Immunol. 130:1473(1983); idem., Cancer Res. 45:4380 (1985); Oseroff et al., Proc Natl.Acad. Sci. USA 83:8744 (1986); idem., Photochem. Photobiol. 46:83(1987); Hasan et al., Prog. Clin. Biol. Res. 288:471 (1989); Tatsuta etal., Lasers Surg. Med. 9:422 (1989); Pelegrin et al., Cancer 67:2529(1991).

For purposes of therapy, the anti-pancreatic cancer antibodies andfragments thereof are administered to a patient in a therapeuticallyeffective amount. An antibody is said to be administered in a“therapeutically effective amount” if the amount administered isphysiologically significant. An agent is physiologically significant ifits presence results in a detectable change in the physiology of arecipient patient.

A diagnostic agent is a molecule or atom, which may be administeredconjugated to an antibody, antibody fragment or fusion protein or atargetable construct, and is useful in diagnosing/detecting a disease bylocating the cells containing the disease-associated antigen. Usefuldiagnostic agents include, but are not limited to, radioisotopes, dyes(such as with the biotin-streptavidin complex), radiopaque materials(e.g., iodine, barium, gallium, and thallium compounds and the like),contrast agents, fluorescent compounds or molecules and enhancing agents(e.g., paramagnetic ions) for magnetic resonance imaging (MRI). U.S.Pat. No. 6,331,175 describes MRI technique and the preparation ofantibodies conjugated to a MRI enhancing agent. Preferably, thediagnostic agents are selected from the group consisting ofradioisotopes for nuclear imaging, endoscopic and intravasculardetection, enhancing agents for use in magnetic resonance imaging or inultrasonography, radiopaque and contrast agents for X-rays and computedtomography, and fluorescent compounds for fluoroscopy, includingendoscopic fluoroscopy. Fluorescent and radioactive agents conjugated toantibodies or used in bispecific, pretargeting methods, are particularlyuseful for endoscopic, intraoperative or intravascular detection of thetargeted antigens associated with diseased tissues or clusters of cells,such as malignant tumors, as disclosed in Goldenberg U.S. Pat. Nos.5,716,595; 6,096,289 and U.S. application Ser. No. 09/348,818, theExamples section of each incorporated herein by reference, particularlywith gamma-, beta- and positron-emitters. Endoscopic applications may beused when there is spread to a structure that allows an endoscope, suchas the colon. Radionuclides useful for positron emission tomographyinclude, but are not limited to: F-18, Mn-51, Mn-52m, Fe-52, Co-55,Cu-62, Cu-64, Ga-68, As-72, Br-75, Br-76, Rb-82m, Sr-83, Y-86, Zr-89,Tc-94m, In-110, I-120, and I-124. Total decay energies of usefulpositron-emitting radionuclides are preferably <2,000 keV, morepreferably under 1,000 keV, and most preferably <700 keV. Radionuclidesuseful as diagnostic agents utilizing gamma-ray detection include, butare not limited to: Cr-51, Co-57, Co-58, Fe-59, Cu-67, Ga-67, Se-75,Ru-97, Tc-99m, In-111, In-114m, I-123, I-125, I-131, Yb-169, Hg-197, andTl-201. Decay energies of useful gamma-ray emitting radionuclides arepreferably 20-2000 keV, more preferably 60-600 keV, and most preferably100-300 keV.

In Vitro Diagnosis

The present invention contemplates the use of anti-pancreatic cancerantibodies to screen biological samples in vitro for the presence of thePAM4 antigen. In such immunoassays, the antibody, antibody fragment orfusion protein may be utilized in liquid phase or bound to a solid-phasecarrier, as described below. For purposes of in vitro diagnosis, anytype of antibody such as murine, chimeric, humanized or human may beutilized, since there is no host immune response to consider.

One example of a screening method for determining whether a biologicalsample contains the PAM4 antigen is the radioimmunoassay (RIA). Forexample, in one form of RIA, the substance under test is mixed with PAM4MAb in the presence of radiolabeled PAM4 antigen. In this method, theconcentration of the test substance will be inversely proportional tothe amount of labeled PAM4 antigen bound to the MAb and directly relatedto the amount of free, labeled PAM4 antigen. Other suitable screeningmethods will be readily apparent to those of skill in the art.

Alternatively, in vitro assays can be performed in which ananti-pancreatic cancer antibody, antibody fragment or fusion protein isbound to a solid-phase carrier. For example, MAbs can be attached to apolymer, such as aminodextran, in order to link the MAb to an insolublesupport such as a polymer-coated bead, a plate or a tube.

Other suitable in vitro assays will be readily apparent to those ofskill in the art. The specific concentrations of detectably labeledantibody and PAM4 antigen, the temperature and time of incubation, aswell as other assay conditions may be varied, depending on variousfactors including the concentration of the PAM4 antigen in the sample,the nature of the sample, and the like. The binding activity of a sampleof anti-pancreatic cancer antibody may be determined according towell-known methods. Those skilled in the art will be able to determineoperative and optimal assay conditions for each determination byemploying routine experimentation.

The presence of the PAM4 antigen in a biological sample can bedetermined using an enzyme-linked immunosorbent assay (ELISA) (e.g.,Gold et al. J Clin Oncol. 24:252-58, 2006). In the direct competitiveELISA, a pure or semipure antigen preparation is bound to a solidsupport that is insoluble in the fluid or cellular extract being testedand a quantity of detectably labeled soluble antibody is added to permitdetection and/or quantitation of the binary complex formed betweensolid-phase antigen and labeled antibody.

In contrast, a “double-determinant” ELISA, also known as a “two-siteELISA” or “sandwich assay,” requires small amounts of antigen and theassay does not require extensive purification of the antigen. Thus, thedouble-determinant ELISA is preferred to the direct competitive ELISAfor the detection of an antigen in a clinical sample. See, for example,the use of the double-determinant ELISA for quantitation of the c-myconcoprotein in biopsy specimens. Field et al., Oncogene 4: 1463 (1989);Spandidos et al., AntiCancer Res. 9: 821 (1989).

In a double-determinant ELISA, a quantity of unlabeled MAb or antibodyfragment (the “capture antibody”) is bound to a solid support, the testsample is brought into contact with the capture antibody, and a quantityof detectably labeled soluble antibody (or antibody fragment) is addedto permit detection and/or quantitation of the ternary complex formedbetween the capture antibody, antigen, and labeled antibody. In thepresent context, an antibody fragment is a portion of an anti-pancreaticcancer MAb that binds to an epitope of the PAM4 antigen. Methods ofperforming a double-determinant ELISA are well-known. See, for example,Field et al., supra, Spandidos et al., supra, and Moore et al.,“Twin-Site ELISAs for fos and myc Oncoproteins Using the AMPAK System,”in METHODS IN MOLECULAR BIOLOGY, VOL. 10, pages 273-281 (The HumanaPress, Inc. 1992).

In the double-determinant ELISA, the soluble antibody or antibodyfragment must bind to a PAM4 epitope that is distinct from the epitoperecognized by the capture antibody. The double-determinant ELISA can beperformed to ascertain whether the PAM4 antigen is present in a biopsysample. Alternatively, the assay can be performed to quantitate theamount of PAM4 antigen that is present in a clinical sample of bodyfluid. The quantitative assay can be performed by including dilutions ofpurified PAM4 antigen.

The anti-pancreatic cancer Mabs, fusion proteins, and fragments thereofalso are suited for the preparation of an assay kit. Such a kit maycomprise a carrier means that is compartmentalized to receive in closeconfinement one or more container means such as vials, tubes and thelike, each of said container means comprising the separate elements ofthe immunoassay.

The subject antibodies, antibody fragments and fusion proteins also canbe used to detect the presence of the PAM4 antigen in tissue sectionsprepared from a histological specimen. Such in situ detection can beused to determine the presence of the PAM4 antigen and to determine thedistribution of the PAM4 antigen in the examined tissue. In situdetection can be accomplished by applying a detectably-labeled antibodyto frozen tissue sections. Studies indicate that the PAM4 antigen ispreserved in paraffin-embedded sections. General techniques of in situdetection are well-known to those of ordinary skill. See, for example,Ponder, “Cell Marking Techniques and Their Application,” in MAMMALIANDEVELOPMENT: A PRACTICAL APPROACH 113-38 Monk (ed.) (IRL Press 1987),and Coligan at pages 5.8.1-5.8.8.

Antibodies, antibody fragments and fusion proteins can be detectablylabeled with any appropriate marker moiety, for example, a radioisotope,an enzyme, a fluorescent label, a dye, a chromagen, a chemiluminescentlabel, a bioluminescent labels or a paramagnetic label.

The marker moiety can be a radioisotope that is detected by such meansas the use of a gamma counter or a scintillation counter or byautoradiography. In a preferred embodiment, the diagnostic conjugate isa gamma-, beta- or a positron-emitting isotope. A marker moiety in thepresent description refers to a molecule that will generate a signalunder predetermined conditions. Examples of marker moieties includeradioisotopes, enzymes, fluorescent labels, chemiluminescent labels,bioluminescent labels and paramagnetic labels.

The binding of marker moieties to anti-pancreatic cancer antibodies canbe accomplished using standard techniques known to the art. Typicalmethodology in this regard is described by Kennedy et al., Clin ChimActa 70:1 (1976), Schurs et al., Clin. Chim. Acta 81: 1 (1977), Shih etal., Int J Cancer 46: 1101 (1990).

The above-described in vitro and in situ detection methods may be usedto assist in the diagnosis or staging of a pathological condition. Forexample, such methods can be used to detect tumors that express the PAM4antigen such as pancreatic cancer.

In Vivo Diagnosis/Detection

Various methods of in vivo diagnostic imaging with radiolabeled MAbs arewell-known. In the technique of immunoscintigraphy, for example,antibodies are labeled with a gamma-emitting radioisotope and introducedinto a patient. A gamma camera is used to detect the location anddistribution of gamma-emitting radioisotopes. See, for example,Srivastava (ed.), RADIOLABELED MONOCLONAL ANTIBODIES FOR IMAGING ANDTHERAPY (Plenum Press 1988), Chase, “Medical Applications ofRadioisotopes,” in REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition,Gennaro et al. (eds.), pp. 624-652 (Mack Publishing Co., 1990), andBrown, “Clinical Use of Monoclonal Antibodies,” in BIOTECHNOLOGY ANDPHARMACY 227-49, Pezzuto et al. (eds.) (Chapman & Hall 1993).

For diagnostic imaging, radioisotopes may be bound to antibody eitherdirectly or indirectly by using an intermediary functional group. Usefulintermediary functional groups include chelators such asethylenediaminetetraacetic acid and diethylenetriaminepentaacetic acid.For example, see Shih et al., supra, and U.S. Pat. No. 5,057,313.

The radiation dose delivered to the patient is maintained at as low alevel as possible through the choice of isotope for the best combinationof minimum half-life, minimum retention in the body, and minimumquantity of isotope which will permit detection and accuratemeasurement. Examples of radioisotopes that can be bound toanti-pancreatic cancer antibody and are appropriate for diagnosticimaging include ^(99m)Tc, ¹¹¹In and ¹⁸F.

The subject antibodies, antibody fragments and fusion proteins also canbe labeled with paramagnetic ions and a variety of radiological contrastagents for purposes of in vivo diagnosis. Contrast agents that areparticularly useful for magnetic resonance imaging comprise gadolinium,manganese, dysprosium, lanthanum, or iron ions. Additional agentsinclude chromium, copper, cobalt, nickel, rhenium, europium, terbium,holmium, or neodymium. Antibodies and fragments thereof can also beconjugated to ultrasound contrast/enhancing agents. For example, oneultrasound contrast agent is a liposome. Also preferred, the ultrasoundcontrast agent is a liposome that is gas filled.

In a preferred embodiment, a bispecific antibody can be conjugated to acontrast agent. For example, the bispecific antibody may comprise morethan one image-enhancing agent for use in ultrasound imaging. In anotherpreferred embodiment, the contrast agent is a liposome. Preferably, theliposome comprises a bivalent DTPA-peptide covalently attached to theoutside surface of the liposome.

Pharmaceutically Suitable Excipients

Additional pharmaceutical methods may be employed to control theduration of action of an anti-pancreatic cancer antibody in atherapeutic application. Control release preparations can be preparedthrough the use of polymers to complex or adsorb the antibody, antibodyfragment or fusion protein. For example, biocompatible polymers includematrices of poly(ethylene-co-vinyl acetate) and matrices of apolyanhydride copolymer of a stearic acid dimer and sebacic acid.Sherwood et al., Bio/Technology 10: 1446 (1992). The rate of release ofan antibody, antibody fragment or fusion protein from such a matrixdepends upon the molecular weight of the antibody, antibody fragment orfusion protein, the amount of antibody within the matrix, and the sizeof dispersed particles. Saltzman et al., Biophys. J. 55: 163 (1989);Sherwood et al., supra. Other solid dosage forms are described in Anselet al., PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5thEdition (Lea & Febiger 1990), and Gennaro (ed.), REMINGTON'SPHARMACEUTICAL SCIENCES, 18th Edition (Mack Publishing Company 1990),and revised editions thereof.

The antibodies, fragments thereof or fusion proteins to be delivered toa subject can comprise one or more pharmaceutically suitable excipients,one or more additional ingredients, or some combination of these. Theantibody can be formulated according to known methods to preparepharmaceutically useful compositions, whereby the immunoconjugate ornaked antibody is combined in a mixture with a pharmaceutically suitableexcipient. Sterile phosphate-buffered saline is one example of apharmaceutically suitable excipient. Other suitable excipients arewell-known to those in the art. See, for example, Ansel et al.,PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5th Edition (Lea& Febiger 1990), and Gennaro (ed.), REMINGTON'S PHARMACEUTICAL SCIENCES,18th Edition (Mack Publishing Company 1990), and revised editionsthereof.

The immunoconjugate or naked antibody can be formulated for intravenousadministration via, for example, bolus injection or continuous infusion.Formulations for injection can be presented in unit dosage form, e.g.,in ampules or in multi-dose containers, with an added preservative. Thecompositions can take such forms as suspensions, solutions or emulsionsin oily or aqueous vehicles, and can contain formulatory agents such assuspending, stabilizing and/or dispersing agents. Alternatively, theactive ingredient can be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

The immunoconjugate, naked antibody, fragment thereof or fusion proteinmay also be administered to a mammal subcutaneously or by otherparenteral routes. In a preferred embodiment, the antibody or fragmentthereof is administered in a dosage of 20 to 2000 milligrams protein perdose. Moreover, the administration may be by continuous infusion or bysingle or multiple boluses. In general, the dosage of an administeredimmunoconjugate, fusion protein or naked antibody for humans will varydepending upon such factors as the patient's age, weight, height, sex,general medical condition and previous medical history. Typically, it isdesirable to provide the recipient with a dosage of immuno conjugate,antibody fusion protein or naked antibody that is in the range of fromabout 1 mg/kg to 20 mg/kg as a single intravenous or infusion, althougha lower or higher dosage also may be administered as circumstancesdictate. This dosage may be repeated as needed, for example, once perweek for four to ten weeks, preferably once per week for eight weeks,and more preferably, once per week for four weeks. It may also be givenless frequently, such as every other week for several months, or morefrequently, such as two- or three-time weekly. The dosage may be giventhrough various parenteral routes, with appropriate adjustment of thedose and schedule.

Kits

Various embodiments may concern kits containing components suitable fortreating or diagnosing diseased tissue in a patient. Exemplary kits maycontain at least one antibody, antigen binding fragment or fusionprotein as described herein. If the composition containing componentsfor administration is not formulated for delivery via the alimentarycanal, such as by oral delivery, a device capable of delivering the kitcomponents through some other route may be included. One type of device,for applications such as parenteral delivery, is a syringe that is usedto inject the composition into the body of a subject. Inhalation devicesmay also be used. In certain embodiments, an anti-pancreatic cancerantibody or antigen binding fragment thereof may be provided in the formof a prefilled syringe or autoinjection pen containing a sterile, liquidformulation or lyophilized preparation of antibody (e.g., Kivitz et al.,Clin. Ther. 2006, 28:1619-29).

The kit components may be packaged together or separated into two ormore containers. In some embodiments, the containers may be vials thatcontain sterile, lyophilized formulations of a composition that aresuitable for reconstitution. A kit may also contain one or more bufferssuitable for reconstitution and/or dilution of other reagents. Othercontainers that may be used include, but are not limited to, a pouch,tray, box, tube, or the like. Kit components may be packaged andmaintained sterilely within the containers. Another component that canbe included is instructions for use of the kit.

EXAMPLES

The examples below are illustrative of embodiments of the currentinvention and are not limiting to the scope of the claims. The examplesdiscuss studies employing an exemplary anti-pancreatic cancer monoclonalantibody (PAM4). Clinical studies with the PAM4 MAb have shown that amajority of pancreatic cancer lesions were targeted in patients andthere was no indication of uptake in normal tissues. Dosimetry indicatedthat it was possible to deliver 10 to 20 cGy/mCi to tumors, with a tumorto red marrow dose ratio of 3:1 to 10:1. The data show that PAM4 isuseful for the treatment of pancreatic cancer.

Example 1 Humanized PAM4 Mab

In preferred embodiments, the claimed methods and compositions utilizethe antibody hPAM4 which is a humanized IgG of the murine PAM4 MAbraised against pancreatic cancer mucin. Humanization of the murine PAM4sequences was utilized to reduce the human antimouse antibody (HAMA)response. To produce the humanized PAM4, murine complementaritydetermining regions (CDR) were transferred from heavy and light variablechains of the mouse immunoglobulin into human framework region (FR)antibody sequences, followed by the replacement of some human FRresidues with their murine counterparts. Humanized monoclonal antibodiesare suitable for use in in vitro and in vivo diagnostic and therapeuticmethods.

Comparison of the variable region framework sequences of the murine PAM4MAb (FIG. 1A and FIG. 1B) to known human antibodies in the Kabatdatabase showed that the FRs of PAM4 Vκ and VH exhibited the highestdegree of sequence homology to that of the human antibodies Walker Vκ(FIG. 3A) and Wil2 VH (FIG. 3B), respectively. Therefore, the Walker Vκand Wil2 VH FRs were selected as the human frameworks into which themurine CDRs for PAM4 Vκ (FIG. 3A) and VH (FIG. 3B) were grafted,respectively. The FR4 sequence of the human antibody, NEWM, however, wasused to replace the Wil2 FR4 sequence for the humanization of the PAM4heavy chain (FIG. 3B). A few amino acid residues in PAM4 FRs that flankthe putative CDRs were maintained in hPAM4 based on the considerationthat these residues have more impact on Ag binding than other FRresidues. These residues were 21M, 47W, 59P, 60A, 85S, 87F, and 100G ofVκ (FIG. 3A) and 27Y, 30P, 38K, 48I, 66K, 67A, and 69L of VH (FIG. 3B).The DNA and amino acid sequences of hPAM4 Vκ (SEQ ID NO:16) and VH (SEQID NO:19) are shown in FIGS. 3A and 3B, respectively.

A modified strategy as described by Leung et al. (Leung et al., 1994))was used to construct the designed Vκ (FIG. 4A) and VH (FIG. 4A) genesfor hPAM4 using a combination of long oligonucleotide syntheses and PCR.For the construction of the hPAM4 VH domain, two long oligonucleotides,hPAM4VHA (173-mer) and hPAM4VHB (173-mer) were synthesized on anautomated DNA synthesizer (Applied Biosystem).

hPAM4VHA represents nt 17 to 189 of the hPAM4 VH domain.

(SEQ ID NO: 20) 5′-AGTCTGGGGC TGAGGTGAAG AAGCCTGGGG CCTCAGTGAAGGTCTCCTGC GAGGCTTCTG GATACACATT CCCTAGCTATGTTTTGCACT GGGTGAAGCA GGCCCCTGGA CAAGGGCTTGAGTGGATTGG ATATATTAAT CCTTACAATG ATGGTACTCA GTACAATGAG AAG-3′

hPAM4VHB represents the minus strand of the hPAM4 VH domaincomplementary to nt 169 to 341.

(SEQ ID NO: 21) 5′-AGGGTTCCCT GGCCCCAGTA AGCAAATCCG TAGCTACCACCGAAGCCTCT TGCACAGTAA TACACGGCCG TGTCGTCAGATCTCAGCCTG CTCAGCTCCA TGTAGGCTGT GTTGATGGACGTGTCCCTGG TCAGTGTGGC CTTGCCTTTG AACTTCTCAT TGTACTGAGT ACC-3′

The 3′-terminal sequences (21 nt residues) of hPAM4VHA and VHB arecomplementary to each other. Under defined PCR condition, the 3′-ends ofhPAM4VHA and VHB anneal to form a short double stranded DNA flanked bythe rest of the long oligonucleotides. Each annealed end serves as aprimer for the transcription of the single stranded DNA, resulting in adouble strand DNA composed of the nt 17 to 341 of hPAM4 VH. This DNA wasfurther amplified in the presence of two short oligonucleotides,hPAM4VHBACK and hPAM4VHFOR to form the full-length hPAM4 VH. Theunderlined portions are restriction sites for subcloning as shown inFIG. 4B.

hPAM4VHBACK (SEQ ID NO: 22)5′-CAG GTG CAG CTG CAG CAG TCT GGG GCT GAG GTG A-3′ hPAM4VHFOR(SEQ ID NO: 23) 5′-TGA GGA GAC GGT GAC CAG GGT TCC CTG GCC CCA-3′

A minimal amount of hPAM4VHA and VHB (determined empirically) wasamplified in the presence of 10 μL of 10×PCR Buffer (500 mM KCl, 100 mMTris HCl buffer, pH 8.3, 15 mM MgCl₂), 2 μmol of hPAM4VHBACK andhPAM4VKFOR, and 2.5 units of Taq DNA polymerase (Perkin Elmer Cetus,Norwalk, Conn.). This reaction mixture was subjected to three cycles ofpolymerase chain reaction (PCR) consisting of denaturation at 94° C. for1 minute, annealing at 45° C. for 1 minute, and polymerization at 72° C.for 1.5 minutes. This procedure was followed by 27 cycles of PCRreaction consisting of denaturation at 94° C. for 1 minute, annealing at55° C. for 1 minute, and polymerization at 72° C. for 1 minute.Double-stranded PCR-amplified product for hPAM4 VH was gel-purified,restriction-digested with PstI and BstEII restriction sites and clonedinto the complementary PstI/BstEII restriction sites of the heavy chainstaging vector, VHpBS2, in which the VH sequence was fully assembledwith the DNA sequence encoding the translation initiation codon and asecretion signal peptide in-frame ligated at the 5′-end and an intronsequence at the 3′-end. VHpBS2 is a modified staging vector of VHpBS(Leung et al., Hybridoma, 13:469, 1994), into which a XhoI restrictionsite was introduced at sixteen bases upstream of the translationinitiation codon to facilitate the next subcloning step. The assembledVH gene was subcloned as a XhoI-BamHI restriction fragment into theexpression vector, pdHL2, which contains the expression cassettes forboth human IgG heavy and light chains under the control of IgH enhancerand MT1 promoter, as well as a mouse d/fr gene as a marker for selectionand amplification. Since the heavy chain region of pdHL2 lacks a BamHIrestriction site, this ligation requires use of a linker to provide abridge between the BamHI site of the variable chain and the HindIII sitepresent in the pdHL2 vector. The resulting expression vectors weredesignated as hPAM4VHpdHL2.

For constructing the full length DNA of the humanized Vκ sequence (FIG.4A), hPAM4VKA (157-mer) and hPAM4VKB (156-mer) were synthesized asdescribed above. hPAM4VKA and VKB were amplified by two shortoligonucleotides hPAM4VKBACK and hPAM4VKFOR as described above.

hPAM4VKA represents nt 16 to 172 of the hPAM4 Vκ domain.

(SEQ ID NO: 24) 5′-CAGTCTCCAT CCTCCCTGTC TGCATCTGTA GGAGACAGAGTCACCATGAC CTGCAGTGCC AGCTCAAGTG TAAGTTCCAGCTACTTGTAC TGGTACCAAC AGAAACCAGG GAAAGCCCCCAAACTCTGGA TTTATAGCAC ATCCAACCTG GCTTCTG-3′

hPAM4VKB represents the minus strand of the hPAM4 Vκ domaincomplementary to nt 153 to 308.

(SEQ ID NO: 25) 5′-GTCCCCCCTC CGAACGTGTA CGGGTACCTA TTCCACTGATGGCAGAAATA AGAGGCAGAA TCTTCAGGTT GCAGACTGCTGATGGTGAGA GTGAAGTCTG TCCCAGATCC ACTGCCACTGAAGCGAGCAG GGACTCCAGA AGCCAGGTTG GATGTG-3′

The 3′-terminal sequences (20 nt residues) of hPAM4VKA and VKB arecomplementary to each other. Under defined PCR condition, the 3′-ends ofhPAM4VKA and VKB anneal to form a short double-stranded DNA flanked bythe rest of the long oligonucleotides. Each annealed end served as aprimer for the transcription of the single stranded DNA, resulting in adouble strand DNA composed of nt 16 to 308 of hPAM4 Vκ. This DNA wasfurther amplified in the presence of two short oligonucleotides,hPAM4VKBACK and hPAM4VKFOR to form the full-length hPAM4 Vκ. Theunderlined portions are restriction sites for subcloning as describedbelow.

hPAM4VKBACK (SEQ ID NO: 26) 5′-GAC ATC CAG CTG ACC CAG TCT CCA TCC TCCCTG-3′ hPAM4VKFOR (SEQ ID NO: 27)5′-TTA GAT CTC CAG TCG TGT CCC CCC TCC GAA CGT-3′

Gel-purified PCR products for hPAM4 Vκ were restriction-digested withPvuII and BglII and cloned into the complementary PvuII/BclI sites ofthe light chain staging vector, VKpBR2. VKpBR2 is a modified stagingvector of VKpBR (Leung et al., Hybridoma, 13:469, 1994), into which aXbaI restriction site was introduced at sixteen bases upstream of thetranslation initiation codon. The assembled Vκ genes were subcloned asXbaI-BamHI restriction fragments into the expression vector containingthe VH sequence, hPAM4VHpdHL2. The resulting expression vectors weredesignated as hPAM4pdHL2.

Approximately 30 μg of hPAM4pdHL2 was linearized by digestion with SalIand transfected into Sp2/0-Ag14 cells by electroporation at 450 V and 25μF. The transfected cells were plated into 96-well plates and incubatedin a CO₂ cell culture incubator for two days and then selected for MTXresistance. Colonies surviving selection emerged in two to three weeksand were screened for human antibody secretion by ELISA assay. Briefly,supernatants (˜100 ul) from the surviving colonies were added into thewells of an ELISA microplate precoated with goat anti-human IgG F(ab′)₂fragment-specific Ab. The plate was incubated for one hour at roomtemperature. Unbound proteins were removed by washing three times withwash buffer (PBS containing 0.05% Tween-20). Horseradishperoxidase-conjugated goat anti-human IgG Fc fragment-specific Ab wasadded to the wells. Following incubation for one hour, a substratesolution (100 μL/well) containing 4 mM o-phenylenediaminedihydrochloride (OPD) and 0.04% H₂O₂ in PBS was added to the wells afterwashing. Color was allowed to develop in the dark for 30 minutes and thereaction was stopped by the addition of 50 μL of 4 N H₂SO₄ solution. Thebound human IgG was measured by reading the absorbance at 490 nm on anELISA reader. Positive cell clones were expanded and hPAM4 was purifiedfrom cell culture supernatant by affinity chromatography on a Protein Acolumn.

The Ag-binding activity of hPAM4 was confirmed by ELISA assay in amicrotiter plate coated with pancreas cancer cell extracts. An ELISAcompetitive binding assay using PAM4-antigen coated plates was developedto assess the Ag-binding affinity of hPAM4 in comparison with that of achimeric PAM4 composed of murine V and human C domains. Constant amountsof the HRP-conjugated cPAM4 mixed with varying concentrations of cPAM4or hPAM4 were added to the coated wells and incubated at roomtemperature for 1-2 h. The amount of HRP-conjugated cPAM4 bound to theCaPan1 Ag was revealed by reading the absorbance at 490 nm after theaddition of a substrate solution containing 4 mM o-phenylenediaminedihydrochloride and 0.04% H₂O₂. As shown by the competition assays inFIG. 5, hPAM4 and cPAM4 antibodies exhibited similar binding activities.

Example 2 Immunohistochemistry Staining Studies

Immunohistochemistry on normal adult tissues showed that the PAM4reactive epitope was restricted to the gastrointestinal tract wherestaining was weak, yet positive (Table 1). Normal pancreatic tissue,including ducts, ductules, acini, and islet cells, were negative forstaining. A PAM4 based enzyme immunoassay with tissue homogenates asantigens generally supported the immunohistology data (Table 2). ThePAM4 epitope was absent from normal pancreas and othernon-gastrointestinal tissues. In neoplastic tissues, PAM4 was reactivewith twenty one out of twenty five (85%) pancreatic cancers (Table 3 andTable 4) and ten out of twenty six colon cancers, but only limitedreactivity with tumors of the stomach, lung, breast, ovary, prostate,liver or kidney (Table 4). PAM4 reactivity appeared to correlate withthe stage of tumor differentiation, with a greater percentage ofstaining observed in well differentiated pancreatic cancers than inmoderately differentiated or poorly differentiated tumors. Generally,poorly differentiated tumors represent less than 10% of all pancreaticcancers.

These studies have shown the PAM4 reactivity and tissue distribution(both normal and cancer) to be unlike that reported for the CA19.9,DUPAN2, SPAN1, Nd2 and B72.3 antibodies and antibodies against the Lewisantigens. Together with crossblocking studies performed with certain ofthese MAbs, the data suggests that the PAM4 MAb recognizes a unique andnovel epitope. When compared to the antigens recognized by the CA19.9,DUPAN2, and anti-Le^(a) antibodies, the PAM4 antigen appears to be morerestricted in its tissue distribution and is reactive with a higherpercentage of pancreatic tumors. Moreover, it gives a greater overallintensity of reaction at equivalent concentrations and is reactive witha higher percentage of cells within the pancreatic tumors. Finally, PAM4was found to be only weakly reactive with three out of twelve chronicpancreatitis specimens, whereas CA19.9 and DUPAN2 were strongly reactivewith all twelve specimens. Although it is recognized that specificity isdependent in part upon the type of assay employed and the range andnumber of tissues examined, the ability of PAM4 to discriminate betweennormal and neoplastic pancreatic tissue, its ability to react with alarge percentage of the cancer specimens, the high intensity of thereactions, and the ability to distinguish between early stage pancreaticcancer and benign conditions such as pancreatitis are importantcharacteristics of this exemplary anti-pancreatic cancer antibody.

TABLE 1 Immunoperoxidase Staining of Normal Adult Tissues with MAb PAM4Staining Tissue Reaction Pancreas (22)^(a) − Ducts − Acini − Islets −Submaxillary gland (2) − Esophagus (2) − Stomach (3) +mucus secretingcells Duodenum (3) +goblet cells Jejunum (3) +goblet cells Ileum (3)+goblet cells Colon (5) +goblet cells Liver (3) − Gallbladder (2) −Bronchus (3) − Lung (3) − Heart (3) − Spleen (3) − Kidney (3) − Bladder(3) − Prostate (2) − Testes (2) − Uterus (2) − Ovary (2) − ^(a)number ofindividual specimens examined in parentheses

TABLE 2 Monoclonal Antibody PAM4 Reactivity with Normal Adult TissueHomogenates by EIA Tissue μg/g tissue^(a) Pancreas 6.4 Esophagus 8.1Stomach 61.3 Duodenum 44.7 Jejunum 60.6 Colon 74.5 Liver 0.0 Gallbladder5.6 Heart 3.7 Spleen 3.4 Kidney 6.6 Bladder 4.9 Thyroid 3.5 Adrenal 1.3Ureter 2.6 Testes 3.9 CaPan1 Pancreatic Tumor 569 ^(a)values are meanfrom two autopsy specimens

TABLE 3 Immunohistochemical Reactivity of Several Monoclonal Antibodieswith Pancreatic Tumors Differentiation PAM4 CA19.9 Le^(a) DUPAN2  1 W+++ − − +++  2 M ++ +++ +++ +  3 M + − + +  4 M +++ +++ +++ +  5 M ++ +− −  6 M + ND ND ND  7 M* +++ +++ +++ +++  8 M + − − +++  9 M ++ + ++ −10 M* ++ ++ ++ +++ 11 M ++ +++ +++ + 12 M ++ + + +++ 13 M + +++ +++ + 14M ++ + + ++ 15 M +++ + + ++ 16 M + + ++ − 17 M − + + − 18 M ++ ++ ++ ++19 M +++ + +++ ++ 20 M + − − − 21 M +++ +++ + ++ 22 P + + + +++ 23 P − −− − 24 P − − − − 25 P − − + − TOTAL 21/25 17/24 18/24 16/24 −: Negative;+: 5-20% of tissue is stained; ++: 21-50% of tissue is stained;+++: >50% of tissue is stained; W, M, P: Well, moderate, or poordifferentiation; *Metastatic tissue; ND: Not Done

TABLE 4 Immunoperoxidase Staining of Neoplastic Tissues with MAb PAM4Cancer Tissue Positive/Total Pancreas 21/25 Colon 10/26 Stomach 1/5 Lung 1/15 Breast  0/30 Ovarian  0/10 Prostate 0/4 Liver  0/10 Kidney 0/4

Example 3 In Vivo Biodistribution and Tumor Targeting of RadiolabeledPAM4

Initial biodistribution studies of PAM4 were carried out in a series offour different xenografted human pancreatic tumors covering the range ofexpected differentiation. Each of the four tumor lines employed, AsPc1,BxPc3, Hs766T and CaPan1, exhibited concentrations of ¹³¹I-PAM4 withinthe tumors (range: 21%-48% ID/g on day three) that were significantly(P<0.01-0.001) higher than concomitantly administered nonspecific,isotype-matched Ag8 antibody (range: 3.6%-9.3% ID/g on day three). Thebiodistribution data were used to estimate potential radiation doses tothe tumor of 12,230; 10,684; 6,835; and 15,843 cGy/mCi of injected doseto AsPc1, BxPc3, Hs766T and CaPan1, respectively. With an actual maximumtolerated dose (MTD) of 0.7 mCi, PAM4 could provide substantial rad doseto each of the xenografted tumor models. In each tumor line the bloodlevels of radiolabeled PAM4 were significantly (P<0.01-0.001) lower thanthe nonspecific Ag8. Potential radiation doses to the blood from PAM4were 1.4-4.4 fold lower than from Ag8. When radiation doses to the tumorfrom PAM4 were normalized to the blood doses from PAM4, the tumorsreceived doses that were 2.2; 3.3; 3.4; and 13.1-fold higher than blood,respectively. Importantly, potential radiation doses to non-tumortissues were minimal.

The biodistribution of PAM4 was compared with an anti-CEA antibody,MN-14, using the CaPan1 tumor model. The concentration of PAM4 withinthe tumor was much greater than MN-14 at early timepoints, yieldingtumor:blood ratios at day three of 12.7±2.3 for PAM4 compared to 2.7±1.9for MN-14. Although PAM4 uptake within the tumor was significantlyhigher than for MN-14 at early timepoints (day one—P<0.001; daythree—P<0.01), dosimetry analyses indicated only a 3.2-fold higher doseto the tumor from PAM4 as compared to MN-14 over the fourteen day studyperiod. This was due to a rapid clearance of PAM4 from the tumor, suchthat at later timepoints similar concentrations of the two antibodieswere present within the tumors. A rapid clearance of PAM4 from the tumorwas also noted in the BxPc3 and Hs766T but not AsPc1 tumor models. Theseobservations were unlike those reported for other anti-mucin antibodies,as for example G9 and B72.3 in colorectal cancer, where each exhibitedlonger retention times as compared to the MN-14 antibody. Results fromstudies on the metabolism of PAM4, indicate that after initial bindingto the tumor cell, antibody is rapidly released, possibly beingcatabolized or being shed as an antigen:antibody complex. The bloodclearance is also very rapid. These data suggest that ¹³¹I may not bethe appropriate choice of isotope for therapeutic applications. Ashort-lived isotope, such as ⁹⁰Y or ¹⁸⁸Re, that can be administeredfrequently may be a more effective reagent.

PAM4 showed no evidence of targeting to normal tissues, except in theCaPan1 tumor model, where a small but statistically significant splenicuptake was observed (range 3.1-7.5% ID/g on day-3). This type of splenictargeting has been observed in the clinical application of theanti-mucin antibodies B72.3 and CC49. Importantly, these studies alsoreported that splenic targeting did not affect tumor uptake of antibodynor did it interfere with interpretation of the nuclear scans. Thesestudies suggested that splenic targeting was not due to crossreactiveantigens in the spleen, nor to binding by Fc receptors, but rather toone or more of the following possibilities: direct targeting of antigentrapped in the spleen, or indirect uptake of antigen:antibody complexesformed either in the blood or released from the tumor site. The latterwould require the presence of immune complexes in the blood. However,these were not observed when specimens as early as five minutes and aslate as seven days were examined by gel filtration (HPLC, GF-250column); radiolabeled antibody eluted as native material. The formerexplanation seems more likely in view of the fact that the CaPan1 tumorproduced large quantities of PAM4-reactive antigen, 100- to 1000-foldhigher than for the other tumor cell lines examined. The lack of splenictargeting by PAM4 in these other tumor lines suggests that thisphenomenon was related to excessive antigen production. Splenictargeting can be overcome by increasing the protein dose to 10 μg fromthe original 2 μg dose. A greater amount of the splenic entrappedantigen presumably was complexed with unlabeled PAM4 rather thanradiolabeled antibody. Increasing the protein dose had no adverse effectupon targeting of PAM4 to the tumor or nontumor tissues. In fact, anincrease of the protein dose to 100 μg more than doubled theconcentration of radiolabeled PAM4 within the CaPan1 tumor.

Example 4 Development of Orthotopic Pancreatic Tumor Model in AthymicNude Mice

In order to resemble the clinical presentation of pancreatic cancer inan animal model more closely, we developed an orthotopic model byinjecting tumor cells directly into the head of the pancreas. OrthotopicCaPan1 tumors grew progressively without overt symptoms until thedevelopment of ascites and death at ten to fourteen weeks. By three tofour weeks post-implantation, animals developed a palpable tumor ofapproximately 0.2 g. Within eight weeks of growth, primary tumors ofapproximately 1.2 g along with metastases to the liver and spleen wereobserved (1-3 metastatic tumors/animal; each tumor<0.1 g). At ten tofourteen weeks seeding of the diaphragm with development of ascites wereevident. Ascites formation and occasional jaundice were usually thefirst overt indications of tumor growth. At this time tumors were quitelarge, 1 to 2 g, and animals had at most only three to four weeks untildeath occurred.

Radiolabeled ¹³¹I-PAM4, administered to animals bearing four week oldorthotopic tumors (approximately 0.2 g) showed specific targeting to theprimary tumor with localization indices of 7.9±3.0 at day one increasingto 22.8±15.3 at day fourteen. No evidence of specific targeting to othertissues was noted. In one case where tumor metastases to the liver andspleen were observed, both metastases were targeted, and had highconcentrations of radiolabeled antibody. In addition, approximately halfof the animals developed a subcutaneous tumor at the incision site. Nosignificant differences were noted in the targeting of orthotopic andsubcutaneous tumors within the same animal, and no significantdifferences were observed in the targeting of orthotopic tumor whetheror not the animal had an additional subcutaneous tumor. The estimatedradiation doses from PAM4 were 6,704 and 1,655 cGy/mCi to the primarytumor and blood, respectively.

Example 5 Experimental Radioimmunotherapy of Pancreatic Cancer

The initial studies on the use of ¹³¹I-PAM4 for therapy were carried outwith the CaPan1 tumor, which was grown as a subcutaneous xenograft inathymic mice. Animals bearing a 0.25 g tumor were administered 350 μCi,¹³¹I-PAM4 in an experiment that also compared the therapeutic effects ofa similar dose of nonspecific Ag8. The MTD for administration of¹³¹I-PAM4 to animals bearing 1 cm³ tumors is 700 μCi. By weeks five andsix, the PAM4 treated animals showed a dramatic regression of tumor, andeven at week twenty seven, five out of eight remained tumor free. Theuntreated, as well as Ag8-treated animals, showed rapid progression oftumor growth although a significant difference was noted between thesetwo control groups. At seven weeks, tumors from the untreated group hadgrown 20.0±14.6-fold from the initial timepoint whereas the¹³¹I-Ag8-treated tumors had grown only 4.9±1.8-fold. At this time point,the PAM4 tumors had regressed to 0.1±0.1-fold of their original size, asignificant difference from both untreated (P<0.001) and nonspecificAg8-treated (P<0.01) animals.

These data show that CaPan1 tumors were sensitive to treatment with¹³¹I-PAM4. The outcome, that is, regression or progression of the tumor,was dependent upon several factors including initial tumor size. Thus,groups of animals bearing CaPan1 tumor burdens of 0.25 g, 0.5 g, 1.0 g,or 2.0 g were treated with a single dose of the 350 μCi ¹³¹I-PAM4. Themajority of animals having tumors of initial size 0.25 g and 0.5 g (nineof ten animals in each group) showed tumor regression or growthinhibition for at least sixteen weeks post treatment. In the 1.0 g tumorgroup five out of seven showed no tumor growth for the sixteen weekperiod and in the 2.0 g tumor group six out of nine showed no tumorgrowth for a period of six weeks before progression occurred. Although asingle 350 μCi dose was not as effective against larger tumors, a singledose may not be the appropriate regimen for large tumors.

Toxicity studies indicate the ability to give multiple cycles ofradioimunotherapy, which may be more effective with a larger tumorburden. Animals bearing CaPan1 tumors averaging 1.0 g, were given eithera single dose of 350 μCi ¹³¹I-PAM4, two doses given at times zero andfour weeks or were left untreated. The untreated group had a meansurvival time of 3.7±1.0 weeks (survival defined as time for tumor toreach 5 cm³). Animals died as early as three weeks, with no animalsurviving past six weeks. A single dose of 350 μCi ¹³¹I-PAM4 produced asignificant increase in the survival time to 18.8±4.2 weeks (P<0.0001).The range of animal deaths extended from weeks thirteen to twenty five.None of the animals were alive at the end of the study period of twentysix weeks.

A significant increase in survival time was observed for the two dosegroup as compared to the single dose group. Half of the animals werealive at the twenty six week timepoint with tumor sizes from 1.0-2.8cm³, and a mean tumor growth rate of 1.6±0.7 fold from initial tumorsize. For those animals that were non-survivors at twenty six weeks, themean survival time (17.7±5.3 weeks) was similar to the single dosegroup.

Therapy studies with PAM4 were also conducted using the orthotopic tumormodel. Groups of animals bearing four week old orthotopic tumors(estimated tumor weight of 0.25 g) were either left untreated or treatedwith a single dose of either 350 μCi ¹³¹I-PAM4 or 350 μCi of¹³¹I-nonspecific Ag8. The untreated animals had a 50% death rate by weekten with no survivors at week fifteen. Animals administered nonspecific¹³¹I-Ag8 at four weeks of tumor growth, showed a 50% death rate at weekseven with no survivors at week fourteen. Although statistically(logrank analysis) there were no differences between these two groups,it is possible that radiation toxicity had occurred in the Ag8 treatedanimals. Radiolabeled PAM4 provided a significant survival advantage(P<0.001) as compared to the untreated or Ag8 treated animals, with 70%survival at sixteen weeks, the end of the experiment. At this time thesurviving animals were sacrificed to determine tumor size. All animalshad tumor with an average weight of 1.2 g, as well as one or two small(<0.1 g) metastases evident in four of the seven animals. At sixteenweeks of growth, these tumors were more representative of aneight-week-old tumor.

Example 6 Combined Modality GEMZAR® Chemotherapy and ¹³¹I-PAM4Experimental Radioimmunotherapy

Initial studies into the combined use of gemcitabine (GEMZAR®) with¹³¹I-PAM4 radioimmunotherapy were performed as a checkerboard array; asingle dose of gemcitabine (0, 100, 200, 500 mg/kg) versus a single doseof ¹³¹I-PAM4 ([MTD=700 μCi] 100%, 75%, 50%, 0% of the MTD). The combinedMTD was found to be 500 mg/kg gemcitabine with 350 μCi ¹³¹I-PAM4 (50%MTD). Toxicity, as measured by loss of body weight, went to the maximumconsidered as nontoxic; that is 20% loss in body weight. Although thecombined treatment protocol was significantly more effective thangemcitabine alone, the treatment was no more effective thanradioimmunotherapy alone. The next studies were performed at a low doseof gemcitabine and radioimmunotherapy to examine if a true synergistictherapeutic effect would be observed. Athymic nude mice bearing tumorsof approximately 1 cm³ (approximately 5% of body weight) wereadministered gemcitabine, 100 mg/kg on days zero, three, six, nine, andtwelve, with 100 μCi of ¹³¹I-PAM4 given on day zero. A therapeuticeffect was observed with statistically significant (P<0.0001) regression(two of five tumors less than 0.1 cm³) and/or growth inhibition of thetumors compared to gemcitabine alone. Thus, at lower dosages oftherapeutic agent, there surprisingly appears to be a synergistic effectof the combination of gemcitabine and radioimmunotherapy. Of additionalnote, in terms of body weight, toxicity was not observed. Thecombination treatment protocol can, if necessary, be delivered inmultiple cycles, with the second treatment cycle beginning in week-four,as was done with the radioimmunotherapy-alone studies described above.

Example 7 Pretargeting with Bispecific cPAM4×734 and ^(99m)Tc- or¹¹¹In-Labeled Peptide Haptens

For imaging of pancreatic cancer using a pretargeted approach weprepared a bispecific F(ab′)₂ antibody (bsMAb) consisting of a chimericPAM4 (cPAM4) Fab′ and a murine 734 (m734) Fab′. The murine 734 antibodyrecognizes an In-DTPA complex. This bsMAb was labeled with ¹²⁵I (7 μCi)and injected into athymic nude mice bearing a human pancreatic cancerxenograft (CaPan1). A non-targeting F(ab′)₂ bsMAb made from chimericrituximab (anti-CD20 monoclonal antibody) and m734, was labeled with¹³¹I and co-injected as a control. At various time-points (4, 24, 36,48, and 72-hours post-injection) mice were necropsied, the tissuesremoved and counted to determine percent-injected dose per gram (%ID/g). There was significantly greater tumor uptake of bsPAM4 at eachtime-point in comparison to the control bs-rituximab (P<0.032 orbetter). Our past experience with this type of pre-targeting systemsuggested that a blood level of less than 1% ID/g was necessary toobtain good tumor:non-tumor ratios. At 36-hours post-administration ofthe bsPAM4 there was 1.10±0.40% ID/g in the blood which fell to0.56±0.08% ID/g at 48 hours post-injection. Tumor uptake at these twotime-points was 6.43±1.50% ID/g and 5.37±2.38% ID/g, respectively. Thesevalues were significantly higher than the control bs-rituximab which had0.65±0.33% ID/g and 0.47±0.19% ID/g in the tumor at 36 and 48 hours,respectively (P<0.018 and P<0.0098). Blood clearance rates, however,were very similar and were not significantly different.

Based on these data a pre-targeting experiment was carried out in CaPan1tumor-bearing mice in which radiolabeled peptide-haptens were injected40-hours post-bsMAb administration. Two peptides, IMP-192 and IMP-156,were used, each containing divalent DTPA for recognition by the 734 MAb,but one had an additional group specific for binding ^(99m)Tc stably(IMP-192). Tumor-bearing mice (tumor volume about 0.30 cm³) wereadministered ¹²⁵I-bsPAM4 (6 μCi) followed 40 hours later by aradiolabeled peptide-hapten (34.5 μCi; 1.5×10⁻¹¹ moles;bsMAb:peptide=10:1). One group of mice received ^(99m)Tc-labeled IMP192while a second group of mice received ¹¹¹In-labeled IMP 156. Controlsfor non-specific targeting included two groups that received¹²⁵I-bs-rituximab prior to administration of radiolabeled peptide andtwo other groups that received ¹¹¹In- or ^(99m)Tc-labeled peptide alone.

Mice were sacrificed at 3 and 24 hours after the administration ofpeptides and the % ID/g determined for the tumor and various tissues.Consistent with our previous findings, there was significantly greaterbsPAM4 in the tumors in comparison to the non-targeting controlbsRituximab, 8.2±3.4% and 0.3±0.08% ID/g, respectively (P<0.0001). Thistranslated into a significantly greatly tumor uptake of ¹¹¹In-IMP 156(20.2±5.5% ID/g vs. 0.9±0.1% ID/g, P<0.0001). There was alsosignificantly greater tumor uptake of ^(99m)Tc-IMP192 in the micepre-targeted with bsPAM4 than in those pre-targeted with bs-rituximab(16.8±4.8% ID/g vs. 1.1±0.2% ID/g, P<0.0005). Tumor uptake of eachpeptide, when administered alone, was significantly less than in thosemice that received the bsPAM4 (0.2±0.05% ID/g and 0.1±0.03% ID/g for^(99m)Tc-IMP 192 and ¹¹¹In-IMP156, P<0.0004 and P<0.0001, respectively).

As with the 3-hour time-point, there was significantly more bsPAM4 inthe tumors at 24 hours post-injection of peptide (64 hours post bsMAbadministration) than bs-rituximab (6.4±2.2% ID/g vs. 0.2±0.09% ID/g,respectively; P<0.0001). At this time-point there was 11.1±3.5% ID/g¹¹¹In-IMP156 and 12.9±4.2% ID/g ^(99m)Tc-IMP192 in the tumors of micepre-targeted with bsPAM4 versus 0.5±0.2% ID/g and 0.4±0.03% ID/g inbs-rituximab pre-targeted tumors (P<0.0008 and P<0.0002, respectively).In the mice that received peptide alone, there was significantly less^(99m)Tc-IMP192 in the tumors (0.06±0.02% ID/g, P<0.0007) and¹¹¹In-IMP156 (0.09±0.02% ID/g, P<0.0002) in comparison to the bsPAM4pre-targeted peptides.

TABLE 5 Tumor:Non-Tumor Tissue Ratios at Early Time-Points Pre-targetedPre-targeted ¹²⁵I-bsPAM4 ¹¹¹In-Peptide ^(99m)Tc-Peptide F(ab′)₂(3-Hours) (3-Hours) (4-Hours) Mean (±STD) Mean (±STD) Mean (±STD) TissueTumor 1.00 0.00 1.00 0.00 1.00 0.00 Liver 36.07 11.74 16.66 7.19 2.340.61 Spleen 33.40 20.62 14.62 9.12 2.15 .074 Kidney 7.79 2.81 8.13 3.331.10 .020 Lung 44.55 12.99 15.75 5.85 1.58 .037 Blood 36.47 8.28 9.935.21 0.47 0.11 Bone 123.24 40.00 — — — — W. Bone 378.00 124.57 — — — —Pancreas 155.55 30.07 73.29 32.85 4.65 1.23 Tumor Wt (g) 0.189 (0.070)0.174 (0.040) 0.179 (0.139) (±STD)

Table 5 presents the tumor:non-tumor ratios (T:NT) of various tissuesfor these groups, each at an early time-point post-administration ofradiolabeled product. It is important to note that at 4-hourspost-administration of bsPAM4×m734 F(ab′)₂, the tumor:blood ratio wasless than 2:1. However, at 3-hours post-administration, the pre-targeted¹¹¹In-IMP156 and ^(99m)Tc-IMP192 had significantly greatertumor:nontumor ratios for all tissues examined and in particulartumor:blood ratios were equal to 36:1 and 9:1, (P<0.001 and P<0.011,respectively). When we examined tumor:blood ratios at the 24-hourtime-point, the pre-targeted ¹¹¹In-IMP 156 and ^(99m)Tc-IMP192 hadsignificantly higher values, 274:1 and 80:1, respectively, versus 4:1for ¹²⁵I-bsPAM4 alone (P<0.0002). These data strongly support theability to utilize this pretargeted bsPAM4 approach with shorthalf-life, high energy radioisotopes that would then deliver highradiation dose to tumor with minimal radiation dose to non-tumortissues.

Example 8 Binding of PAM4 Antibodies to Transfected Cell Lines

Transfected Pancreatic Cells

PanC1 human pancreatic adenocarinoma cells that do not express the MUC-1mucin were transfected with MUC-1 encoding cDNA as disclosed in Hudsonet al. (Amer. J. Pathol. 148, 3:951-60, 1996) and obtained from Dr. M.A. Hollingsworth (Univ. of Nebraska Medical Center, Omaha, Nebr.). TheMUC-1 cDNAs encoded either 30 tandem repeat (30TR) or 42 tandem repeat(42TR) versions of MUC-1 (Hudson et al., 1996; Lidell et al, FEBS J.275:481-89, 2008). The MUC-1 sequences were identical other than in thenumber of tandem repeat sequences encoded.

PanC1 cells transfected with either 30TR or 42TR MUC-1 or controlvectors (no insert or reversed insert) or untransfected PanC1 cells wereexamined for reactivity with PAM4 antibody by enzyme immunoassay of thesupernatants from cell culture. Neither the untransfected PanC1 cellsnor the control transfected PanC1 cells produced detectable levels ofPAM4-reactive mucins by immunoassay (not shown). However, both the 30TRand 42TR MUC-1 transfected cells were highly reactive with PAM4 antibody(not shown).

The transfected PanC1 cells were reexamined using a more sensitiveimmunoassay. Briefly, cells were grown in T75 flasks until they reachedapproximately 80%-90% confluency (˜4-5 days from initial seeding). Atthis time, the spent-media were collected, centrifuged at high speed,and used for quantitation of PAM4-reactive mucin by enzyme immunoassay.The cells were also collected and counted. Both the Panel parent cellline originating from Dr. Hollingsworth and a separate Panel cell lineobtained from the American Type Culture Collection (Manassas, Va.), aswell as the vector control, produced low, but detectable quantities ofPAM4-reactive mucin (0.87±0.17, 0.54±0.17 and 0.02±0.02 μg/mL/10⁶ cells,respectively), whereas the 30TR-MUC-1 gene transfected cells produced14.17±2.22 μg/mL/10⁶ cells (P<0.0003 or better for comparison of30TR-MUC-1 gene transfected media compared to all other samples).

It has been reported that transfection of PanC1 cells with MUC-1 cDNA,in addition to increasing expression of MUC-1 by the transfected PanC1cells, also has secondary effects on cell protein expression, such asincreasing levels of cytokeratins 8 and 18 in the transfected cells(Hudson et al., 1996, Abstract), but only in cells transfected with thelarger (42TR) MUC-1 cDNA (Hudson et al. 1996, pg. 956, col. 1, 3^(rd)paragraph).

Transfected Kidney Cells

MUC-1 gene-transfected HEK-293 (human embryonic kidney cells) producedMUC-1 that was reactive with the MA5 monoclonal antibody, but that wasnot reactive with PAM4 (not shown). However, as discussed above,MUC-1-transfected PanC1 cells that express very low levels of endogenousMUC-1 synthesized MUC-1 that was strongly reactive with PAM4 andnon-reactive with MAb-MA5. Heterologous sandwich immunoassays (PAM4→MA5and MA5→PAM4 capture/probe) did not function to produce a signal withsupernatants from several cell lines. Use of a polyclonal anti-mucinantiserum as probe with the PAM4 or MA5 MAbs as capture reagents didprovide effective immunoassays. Cross-blocking of these two MAbs withintheir respective immunoassays using the polyclonal as the probesuggested these MAbs were reactive with independent epitopes.

The data suggest that the PAM4 and MA5 epitopes are not co-expressedwithin the same antigenic molecule, and that while the PanC1 cell linemay possess biosynthetic processes that create the PAM4-epitope, theHEK-293 cells do not. Differences in post-translational modification ofthe MUC-1 protein core (expression/activity of specificglycosyltransferases) may be responsible for these findings.

Example 9 Effects of Reagent Treatment on Immunoreactivity of PAM4Antigen

Treatment of pancreatic mucin PAM4 antigen with DTT (15 min at roomtemp), completely abolished reactivity with PAM4 (DTT-EC₅₀, 0.60±0.00μM). The only cysteines (cystine bridges) within MUC-1 are presentwithin the transmembrane domain and should not be accessible to DTT. Thesecreted form of MUC-1 does not contain the transmembrane domain andtherefore has no intramolecular cystine bridges. Data from periodateoxidation treatment of PAM4 antigen with 0.05 M sodium periodate for 2hrs at room temperature yielded 40% loss of immunoreactivity with PAM4antibody (not shown). Further periodate studies have shown as high as a60% loss of immunoreactivity with PAM4 antibody (not shown). The resultsof periodate and DTT studies suggest that the PAM4 epitope isconformationally dependent upon some minimal form of glycosylation, andmay be affected by intermolecular disulfide bond formation.

Example 10 Distribution and Cross-Reactivity of the PAM4 Antigen

The expression of the PAM4-epitope within PanINs is atypical for MUC-1.It is similar to the expression reported for MUC-5ac as detected by thecommercially available MAb-CLH2-2. However, an attempted sandwichimmunoassay with PAM4 capture and MAb-CLH2-2 as probe gave negativeresults. Although this possibly suggests the PAM4 and CLH2-2 epitopesmay overlap and thus block each other, the CLH2-2 was reported to bereactive with 42/66 (64%) gastric carcinomas whereas the PAM4 MAb showedreactivity with only 6/40 (15%) of gastric carcinomas and, of these,only in focal reactivity.

Use of the commercially available 45M1, an anti-MUC-5ac MAb, as a probereagent in EIA (with PAM4 as capture) provided positive results,indicating that the two epitopes may be present on the same antigenicmolecule. Blocking studies (either direction) indicated that theepitopes bound by 45M1 and PAM4 are in fact two distinct epitopes, as noblocking was observed. Labeling of tissue microarrays consisting ofcores from invasive pancreatic carcinoma has demonstrated significantdifferences for expression of the 45M1 and PAM4 epitopes in individualpatient specimens. Of 28 specimens, concordance was observed in only 17cases (61%). PAM4 was reactive with 24/28 cases (86%) while 45M1 wasreactive with 13/28 (46%) cases (not shown).

It is possible that in the studies above (Example 8) regarding MUC-1gene transfection, expression of MUC-1 may have upregulated expressionof another mucin, or affected exposure of the PAM4-epitope in some othermanner. The results of periodate studies are consistent withglycosylation as a factor in PAM4 antigen immunoreactivity with the PAM4antibody. Thus, results of studies with apomucins may not be definitivefor antigen determination.

Although based on EIA capture, the PAM4 antibody appears to bind to thesame antigenic protein as the 45M1 anti-MUC-5ac MAb, it is noted thatMUC-5ac is not specific to pancreas cancer and it is found in a numberof normal tissues (other than the gastric mucosa with which PAM4 isreactive). For example, MUC-5ac is found in normal lung, colon and othertissues. PAM4 antibody does not bind to normal lung tissues, except asindicated above in few samples and to a limited or minimal amount.

With respect to the effects of DTT and periodate, it is probable thatthe peptide core disulfide bridges are identical no matter what tissueproduces the protein. A specific amino acid sequence should fold in aspecific manner, independent of the tissue source. However,glycosylation patterns may differ dependent upon tissue source.

Example 11 Phage Display Peptide Binding of PAM4 Antibody

PAM4 antibody binding was examined with two different phage displaypeptide libraries. The'first was a linear peptide library consisting of12 amino acid sequences and the second was a cyclic peptide consistingof 7 amino acid sequences cyclized by a disulfide bridge. We panned theindividual libraries alternately against hPAM4 and hLL2 (negativeselection with anti-CD22 antibody) for a combined total of 4 rounds, andthen screened the phage displayed peptide for reactivity with both hPAM4and mPAM4 with little to no reactivity against hLL2. Phage binding in anon-specific manner (i.e., binding to epratuzumab [hLL2]) werediscarded.

For the linear phage-displayed peptide, the sequence WTWNITKAYPLP (SEQID NO:29) was identified 30 times (in 35 sequenced phage), each of whichwere shown to have reactivity with PAM4 antibodies. A mutationalanalysis was conducted in which a library based on this sequence andhaving 7.5% degeneracy at each position, was constructed, panned andscreened as before. Variability was noted in the 19 obtained peptidesequences that were positive for PAM4 binding with 7 being identical tothe parental sequence, 5 having the sequence WTWNITKEYPQP (SEQ ID NO:31)and the rest being uniquely present. Table 6 shows the results of thismutational analysis. The upper row lists the sequences identified andthe lower row lists the frequency with which each of the amino acids wasidentified in that position. The parent sequence is most frequent (bold)with the next highest variation a substitution of E for A at position 8and a substitution of Q for L at position 11. It does not appear thatthese substitutions had any great effect upon immunoreactivity.

TABLE 6 Phage Display Amino Acid Sequence Variation with Linear Peptide Binding to PAM4 Antibody (SEQ ID NO: 60) W T W N I TK A Y P L P D R R E T R Q T T I N R M G F C C number of 19 19 19 18 1917 14 10 18 17 11 19 occurrences  1  2  1  5  1  2  5 (out of 19  2  1 1 sequences  1  1  1 analyzed)  1  1  1  1

Results with the phage displayed cyclic library were significantlydifferent from the linear library (Table 7). The sequence ACPEWWGTTC(SEQ ID NO:30) was present in 33 of 35 peptide sequences examined.Analysis of the cyclic library presented the following results(positions with an asterick were invariant and not subject to selectivepressure in the library).

TABLE 7 Phage Display Amino Acid Sequence Variation with Linear Peptide Binding to PAM4 Antibody (SEQ ID NO: 61) A C P E W WG T T C Y S G M S S Q P number of * * 33 35 35 35 34 29 28 * occurrences 2  1  5  4 (out of 19  1  1 sequences  1 analyzed)  1

The two cysteines (at positions 2 and 10) formed a disulfide bridge.Substitution of T at position 9 with any amino acid greatly affectedimmunoreactivity. The sequence GTTGTTC (SEQ ID NO:32) is present withinthe MUC-5ac protein towards the amino terminus as compared to the cyclicpeptide sequence shown above, which shows homology at the C-terminal endof the consensus peptide sequence. However, the cyclic peptide onlyshowed approximately 10% of the immunoreactivity of the linear sequencewith the PAM4 antibody. Both linear and cyclic consensus sequences areassociated with a cysteine, which may or may not relate to the effect ofDTT on PAM4 antigen immunoreactivity.

The results reported herein indicate that the PAM4 epitope is dependentupon a specific conformation which may be produced by disulfide bridges,as well as a specific glycosylation pattern.

Example 12 Immunohistology of Pancreatic Cancer in a PancreatitisSpecimen

Several pathologic conditions predispose patients to the development ofpancreatic carcinoma, such as pancreatitis, diabetes, smoking andothers. Within this pre-selected group of patients, screening measuresare particularly important for the early detection of pancreaticneoplasia. We examined 9 specimens of chronic pancreatitis tissue frompatients having primary diagnosis of this disease. We employed ananti-CD74 MAb, LL1, as an indicator of inflammatory infiltrate, andMAb-MA5 as a positive control for pancreatic ductal and acinar cells.Whereas the two control MAbs provided immunohistologic evidenceconsistent with pancreatitis, in no instance did PAM4 react withinflamed pancreatic tissue. However, in one case, a moderatelydifferentiated pancreatic adenocarcinoma was also present within thetissue specimen. PAM4 gave an intense stain of the neoplastic cellswithin this tumor. In a second case, while the inflamed tissue wasnegative with PAM4, a small PanIN precursor lesion was identified thatwas labeled with PAM4. Labeling of the PanIN within this latter specimenis consistent with early detection of pancreatic neoplasia in a patientdiagnosed with a non-malignant disease. These results show thatdetection and/or diagnosis using the PAM4 antibody may be performed withhigh sensitivity and selectivity for pancreatic neoplasia against abackground of benign pancreatic tissues.

Example 13 Therapy of a Patient with Inoperable and MetastaticPancreatic Carcinoma

Patient 118-001, CWG, is a 63-year-old man with Stage-IV pancreaticadenocarcinoma with multiple liver metastases, diagnosed in November of2007. He agreed to undertake combined radioimmunotherapy and gemcitabinechemotherapy as a first treatment strategy, and was then given a firsttherapy cycle of 6.5 mCi/m² of ⁹⁰Y-hPAM4, combined with 200 mg/m²gemcitabine, whereby the gemcitabine was given once weekly on weeks 1-4and ⁹⁰Y-hPAM4 was given once-weekly on weeks 2-4 (3 doses). Two monthslater, the same therapy cycle was repeated, because no major toxicitieswere noted after the first cycle. Already 4 weeks after the firsttherapy cycle, CT evidence of a reduction in the diameters of theprimary tumor and 2 of the 3 liver metastases surprisingly was noted,and this was consistent with significant decreases in the SUV values ofFDG-PET scans, with 3 of the 4 tumors returning to normal background SUVlevels at this time (FIG. 6 and FIG. 7). The patient's pre-therapyCA-19.9 level of 1,297 dropped to a low level of 77, further supportiveof the therapy being effective. Table 8 shows the effects of combinedradioimmunotherapy with ⁹⁰Y-hPAM4 and gemcitabine chemotherapy in thispatient. It was surprising and unexpected that such low doses of theradionuclide conjugated to the antibody combined with such low,nontoxic, doses of gemcitabine showed such antitumor activity even afteronly a single course of this therapy.

TABLE 8 Effects of Combined Radioimmunotherapy with ⁹⁰Y-hPAM4 andGemcitabine Chemotherapy in Metastatic Pancreatic Carcinoma BaselineLongest 4 wk post-Tx Baseline Tumor Diameter Longest PET 4 wk post-TxLocation (cm) Diameter (cm) (SUV) PET (SUV) Pancreatic tail 4.5 4.3 9.24.2 (primary) L hepatic met 1.9 1.9 4.1 background R post hepatic 1.71.6 3.7 background met R central hepatic 1.9 1.2 3.2 background met

Example 14 Therapy of a Patient with Inoperable Metastatic PancreaticCarcinoma

A 56-year-old male with extensive, inoperable adenocarcinoma of thepancreas, with several liver metastases ranging from 1 to 4 cm indiameter, substantial weight loss (30 lbs of weight or more), mildjaundice, lethargy and weakness, as well as abdominal pains requiringmedication, is given 4 weekly infusions of gemcitabine at doses of 200mg/m² each. On the last three gemcitabine infusions, ⁹⁰Y-DOTA-hPAM4radiolabeled humanized antibody is administered at a dose of 10 mCi/m²of ⁹⁰Y and 20 mg antibody protein, in a two-hour i.v. infusion. Twoweeks later, the patient is given a course of gemcitabine chemotherapyconsisting of 3 weekly doses of 600 mg/m² by i.v. infusion. The patientis then evaluated 4 weeks later, and has a mild leukopenia (grade-2), noother major blood or enzyme changes over baseline, but shows animprovement in the blood CA19.9 titer from 5,700 to 1,200 and a decreasein jaundice, with an overall subjective improvement. This follows 3weeks later with a repeat of the cycle of lower-dose gemcitabine (weeklyx 4), with 3 doses of ⁹⁰Y-DOTA-hPAM4. Four weeks later, the patient isreevaluated, and the CT and PET scans confirm an approximately 40%reduction of total tumor mass (primary cancer and metastases), with afurther reduction of the CA19.9 titer to 870. The patient regainsappetite and activity, and is able to return to more usual dailyactivities without the need for pain medication. He gains 12 lbs afterbeginning this experimental therapy. A repeat of the scans and bloodvalues indicates that this response is maintained 6 weeks later.

Example 15 Preparation of Dock-and-Lock (DNL) Constructs forPretargeting

DDD and AD Fusion Proteins

The DNL technique can be used to make dimers, trimers, tetramers,hexamers, etc. comprising virtually any antibodies or fragments thereofor other effector moieties. For certain preferred embodiments, IgGantibodies or Fab antibody fragments may be produced as fusion proteinscontaining either a dimerization and docking domain (DDD) or anchoringdomain (AD) sequence. Although in preferred embodiments the DDD and ADmoieties are produced as fusion proteins, the skilled artisan willrealize that other methods of conjugation, such as chemicalcross-linking, may be utilized within the scope of the claimed methodsand compositions.

Bispecific antibodies may be formed by combining a Fab-DDD fusionprotein of a first antibody with a Fab-AD fusion protein of a secondantibody. Alternatively, constructs may be made that combine IgG-ADfusion proteins with Fab-DDD fusion proteins. The technique is notlimiting and any protein or peptide of use may be produced as an AD orDDD fusion protein for incorporation into a DNL construct. Wherechemical cross-linking is utilized, the AD and DDD conjugates are notlimited to proteins or peptides and may comprise any molecule that maybe cross-linked to an AD or DDD sequence using any cross-linkingtechnique known in the art. In certain exemplary embodiments, apolyethylene glycol (PEG) or other polymeric moiety may be incorporatedinto a DNL construct, as described in further detail below.

For pretargeting applications, an antibody or fragment containing abinding site for an antigen associated with a diseased tissue, such as atumor-associated antigen (TAA), may be combined with a second antibodyor fragment that binds a hapten on a targetable construct, to which atherapeutic and/or diagnostic agent is attached. The DNL-basedbispecific antibody is administered to a subject, circulating antibodyis allowed to clear from the blood and localize to target tissue, andthe conjugated targetable construct is added and binds to the localizedantibody for diagnosis or therapy.

Independent transgenic cell lines may be developed for each Fab or IgGfusion protein. Once produced, the modules can be purified if desired ormaintained in the cell culture supernatant fluid. Following production,any DDD-fusion protein module can be combined with any AD-fusion proteinmodule to generate a bispecific DNL construct. For different types ofconstructs, different AD or DDD sequences may be utilized. Exemplary DDDand AD sequences are provided below.

DDD1: (SEQ ID NO: 33) SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA DDD2:(SEQ ID NO: 34) CGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA  AD1:(SEQ ID NO: 35) QIEYLAKQIVDNAIQQA AD2: (SEQ ID NO: 36)CGQIEYLAKQIVDNAIQQAGC

Expression Vectors

The plasmid vector pdHL2 has been used to produce a number of antibodiesand antibody-based constructs. See Gillies et al., J Immunol Methods(1989), 125:191-202; Losman et al., Cancer (Phila) (1997), 80:2660-6.The di-cistronic mammalian expression vector directs the synthesis ofthe heavy and light chains of IgG. The vector sequences are mostlyidentical for many different IgG-pdHL2 constructs, with the onlydifferences existing in the variable domain (VH and VL) sequences. Usingmolecular biology tools known to those skilled in the art, these IgGexpression vectors can be converted into Fab-DDD or Fab-AD expressionvectors. To generate Fab-DDD expression vectors, the coding sequencesfor the hinge, CH2 and CH3 domains of the heavy chain are replaced witha sequence encoding the first 4 residues of the hinge, a 14 residueGly-Ser linker and the first 44 residues of human RIIα (referred to asDDD1). To generate Fab-AD expression vectors, the sequences for thehinge, CH2 and CH3 domains of IgG are replaced with a sequence encodingthe first 4 residues of the hinge, a 15 residue Gly-Ser linker and a 17residue synthetic AD called AKAP-IS (referred to as AD 1), which wasgenerated using bioinformatics and peptide array technology and shown tobind RIIα dimers with a very high affinity (0.4 nM). See Alto, et al.Proc. Natl. Acad. Sci., U.S.A (2003), 100:4445-50.

Two shuttle vectors were designed to facilitate the conversion ofIgG-pdHL2 vectors to either Fab-DDD 1 or Fab-AD 1 expression vectors, asdescribed below.

Preparation of CH₁

The CH1 domain was amplified by PCR using the pdHL2 plasmid vector as atemplate. The left PCR primer consisted of the upstream (5′) end of theCH1 domain and a SacII restriction endonuclease site, which is 5′ of theCH1 coding sequence. The right primer consisted of the sequence codingfor the first 4 residues of the hinge (PKSC) followed by four glycinesand a serine, with the final two codons (GS) comprising a Bam HIrestriction site. The 410 bp PCR amplimer was cloned into the PGEMT® PCRcloning vector (PROMEGA®, Inc.) and clones were screened for inserts inthe T7 (5′) orientation.

Construction of (G₄S)₂DDD1 ((G₄S)₂ Disclosed as SEQ ID NO:37)

A duplex oligonucleotide, designated (G₄S)₂DDD1 ((G₄S)₂ disclosed as SEQID NO:37), was synthesized by Sigma GENOSYS® (Haverhill, UK) to code forthe amino acid sequence of DDD1 preceded by 11 residues of the linkerpeptide, with the first two codons comprising a BamHI restriction site.A stop codon and an EagI restriction site are appended to the 3′ end.The encoded polypeptide sequence is shown below.

(SEQ ID NO: 38) GSGGGGSGGGGSHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTR LREARA

Two oligonucleotides, designated RIIA1-44 top and RIIA1-44 bottom, thatoverlap by 30 base pairs on their 3′ ends, were synthesized (SigmaGENOSYS®) and combined to comprise the central 154 base pairs of the 174bp DDD 1 sequence. The oligonucleotides were annealed and subjected to aprimer extension reaction with Taq polymerase. Following primerextension, the duplex was amplified by PCR. The amplimer was cloned intoPGEMT® and screened for inserts in the T7 (5′) orientation.

Construction of (G₄S)₂ AD1 ((G₄S)₂ Disclosed as SEQ ID NO:37)

A duplex oligonucleotide, designated (G₄S)₂-AD1 ((G₄S)₂ disclosed as SEQID NO:37), was synthesized (Sigma GENOSYS®) to code for the amino acidsequence of AD1 preceded by 11 residues of the linker peptide with thefirst two codons comprising a BamHI restriction site. A stop codon andan EagI restriction site are appended to the 3′ end. The encodedpolypeptide sequence is shown below.

(SEQ ID NO: 39) GSGGGGSGGGGSQIEYLAKQIVDNAIQQA

Two complimentary overlapping oligonucleotides encoding the abovepeptide sequence, designated AKAP-IS Top and AKAP-IS Bottom, weresynthesized and annealed. The duplex was amplified by PCR. The amplimerwas cloned into the PGEMT® vector and screened for inserts in the T7(5′) orientation.

Ligating DDD1 with CH1

A 190 bp fragment encoding the DDD1 sequence was excised from PGEMT®with BamHI and NotI restriction enzymes and then ligated into the samesites in CH1-PGEMT® to generate the shuttle vector CH1-DDD1-PGEMT®.

Ligating AD1 with CH1

A 110 bp fragment containing the AD1 sequence was excised from PGEMT®with BamHI and NotI and then ligated into the same sites in CH1-PGEMT®to generate the shuttle vector CH1-AD1-PGEMT®.

Cloning CH1-DDD1 or CH1 AD1 into pdHL2-Based Vectors

With this modular design either CH1-DDD1 or CH1-AD1 can be incorporatedinto any IgG construct in the pdHL2 vector. The entire heavy chainconstant domain is replaced with one of the above constructs by removingthe SacII/EagI restriction fragment (CH1-CH3) from pdHL2 and replacingit with the SacII/EagI fragment of CH1-DDD1 or CH1-AD1, which is excisedfrom the respective pGemT shuttle vector.

Construction of h679-Fd-AD1-pdHL2

h679-Fd-AD1-pdHL2 is an expression vector for production of h679 Fabwith AD1 coupled to the carboxyl terminal end of the CH1 domain of theFd via a flexible Gly/Ser peptide spacer composed of 14 amino acidresidues. A pdHL2-based vector containing the variable domains of h679was converted to h679-Fd-AD1-pdHL2 by replacement of the SacII/EagIfragment with the CH1-AD1 fragment, which was excised from theCH1-AD1-SV3 shuttle vector with SacII and EagI.

Construction of C-DDD1-Fd-hMN-14-pdHL2

C-DDD1-Fd-hMN-14-pdHL2 is an expression vector for production of astable dimer that comprises two copies of a fusion proteinC-DDD1-Fab-hMN-14, in which DDD1 is linked to hMN-14 Fab at the carboxylterminus of CH1 via a flexible peptide spacer. The plasmid vectorhMN-14(I)-pdHL2, which has been used to produce hMN-14 IgG, wasconverted to C-DDD1-Fd-hMN-14-pdHL2 by digestion with SacII and EagIrestriction endonucleases to remove the CH1-CH3 domains and insertion ofthe CH1-DDD1 fragment, which was excised from the CH1-DDD1-SV3 shuttlevector with SacII and EagI.

The same technique has been utilized to produce plasmids for Fabexpression of a wide variety of known antibodies, such as hLL1, hLL2,hPAM4, hR1, hRS7, hMN-14, hMN-15, hA19, hA20 and many others. Generally,the antibody variable region coding sequences were present in a pdHL2expression vector and the expression vector was converted for productionof an AD- or DDD-fusion protein as described above. The AD- andDDD-fusion proteins comprising a Fab fragment of any of such antibodiesmay be combined, in an approximate ratio of two DDD-fusion proteins perone AD-fusion protein, to generate a trimeric DNL construct comprisingtwo Fab fragments of a first antibody and one Fab fragment of a secondantibody.

C-DDD2-Fd-hMN-14-pdHL2

C-DDD2-Fd-hMN-14-pdHL2 is an expression vector for production ofC-DDD2-Fab-hMN-14, which possesses a dimerization and docking domainsequence of DDD2 appended to the carboxyl terminus of the Fd of hMN-14via a 14 amino acid residue Gly/Ser peptide linker. The fusion proteinsecreted is composed of two identical copies of hMN-14 Fab held togetherby non-covalent interaction of the DDD2 domains.

The expression vector was engineered as follows. Two overlapping,complimentary oligonucleotides, which comprise the coding sequence forpart of the linker peptide (GGGGSGGGCG, SEQ ID NO:40) and residues 1-13of DDD2, were made synthetically. The oligonucleotides were annealed andphosphorylated with T4 PNK, resulting in overhangs on the 5′ and 3′ endsthat are compatible for ligation with DNA digested with the restrictionendonucleases BamHI and PstI, respectively.

The duplex DNA was ligated with the shuttle vector CH1-DDD1-PGEMT®,which was prepared by digestion with BamHI and PstI, to generate theshuttle vector CH1-DDD2-PGEMT®. A 507 bp fragment was excised fromCH1-DDD2-PGEMT® with SacII and EagI and ligated with the IgG expressionvector hMN-14(I)-pdHL2, which was prepared by digestion with SacII andEagI. The final expression construct was designatedC-DDD2-Fd-hMN-14-pdHL2. Similar techniques have been utilized togenerated DDD2-fusion proteins of the Fab fragments of a number ofdifferent humanized antibodies.

h679-Fd-AD2-pdHL2

h679-Fab-AD2, was designed to pair as B to C-DDD2-Fab-hMN-14 as A.h679-Fd-AD2-pdHL2 is an expression vector for the production ofh679-Fab-AD2, which possesses an anchoring domain sequence of AD2appended to the carboxyl terminal end of the CH1 domain via a 14 aminoacid residue Gly/Ser peptide linker. AD2 has one cysteine residuepreceding and another one following the anchor domain sequence of AD1.

The expression vector was engineered as follows. Two overlapping,complimentary oligonucleotides (AD2 Top and AD2 Bottom), which comprisethe coding sequence for AD2 and part of the linker sequence, were madesynthetically. The oligonucleotides were annealed and phosphorylatedwith T4 PNK, resulting in overhangs on the 5′ and 3′ ends that arecompatible for ligation with DNA digested with the restrictionendonucleases BamHI and SpeI, respectively.

The duplex DNA was ligated into the shuttle vector CH1-AD1-PGEMT®, whichwas prepared by digestion with BamHI and SpeI, to generate the shuttlevector CH1-AD2-PGEMT®. A 429 base pair fragment containing CH1 and AD2coding sequences was excised from the shuttle vector with SacII and EagIrestriction enzymes and ligated into h679-pdHL2 vector that prepared bydigestion with those same enzymes. The final expression vector ish679-Fd-AD2-pdHL2.

Generation of TF2 DNL Pretargeting Construct

A trimeric DNL construct designated TF2 was obtained by reactingC-DDD2-Fab-hMN-14 with h679-Fab-AD2. A pilot batch of TF2 was generatedwith >90% yield as follows. Protein L-purified C-DDD2-Fab-hMN-14 (200mg) was mixed with h679-Fab-AD2 (60 mg) at a 1.4:1 molar ratio. Thetotal protein concentration was 1.5 mg/ml in PBS containing 1 mM EDTA.Subsequent steps involved TCEP reduction, HIC chromatography, DMSOoxidation, and IMP 291 affinity chromatography. Before the addition ofTCEP, SE-HPLC did not show any evidence of a₂b formation. Addition of 5mM TCEP rapidly resulted in the formation of a₂b complex consistent witha 157 kDa protein expected for the binary structure. TF2 was purified tonear homogeneity by IMP 291 affinity chromatography (not shown). IMP 291is a synthetic peptide containing the HSG hapten to which the 679 Fabbinds (Rossi et al., 2005, Clin Cancer Res 11:7122s-29s). SE-HPLCanalysis of the IMP 291 unbound fraction demonstrated the removal of a₄,a₂ and free kappa chains from the product (not shown).

Non-reducing SDS-PAGE analysis demonstrated that the majority of TF2exists as a large, covalent structure with a relative mobility near thatof IgG (not shown). The additional bands suggest that disulfideformation is incomplete under the experimental conditions (not shown).Reducing SDS-PAGE shows that any additional bands apparent in thenon-reducing gel are product-related (not shown), as only bandsrepresenting the constituent polypeptides of TF2 were evident (notshown). However, the relative mobilities of each of the fourpolypeptides were too close to be resolved. MALDI-TOF mass spectrometry(not shown) revealed a single peak of 156,434 Da, which is within 99.5%of the calculated mass (157,319 Da) of TF2.

The functionality of TF2 was determined by BIACORE® assay. TF2,C-DDD1-hMN-14+h679-AD1 (used as a control sample of noncovalent a₁bcomplex), or C-DDD2-hMN-14+h679-AD2 (used as a control sample ofunreduced a₂ and b components) were diluted to 1 μg/ml (total protein)and passed over a sensorchip immobilized with HSG. The response for TF2was approximately two-fold that of the two control samples, indicatingthat only the h679-Fab-AD component in the control samples would bind toand remain on the sensorchip. Subsequent injections of WI2 IgG, ananti-idiotype antibody for hMN-14, demonstrated that only TF2 had aDDD-Fab-hMN-14 component that was tightly associated with h679-Fab-AD asindicated by an additional signal response. The additional increase ofresponse units resulting from the binding of WI2 to TF2 immobilized onthe sensorchip corresponded to two fully functional binding sites, eachcontributed by one subunit of C-DDD2-Fab-hMN-14. This was confirmed bythe ability of TF2 to bind two Fab fragments of WI2 (not shown).

Production of TF10 Bispecific Antibody for Pretargeting

A similar protocol was used to generate a trimeric TF10 DNL construct,comprising two copies of a C-DDD2-Fab-hPAM4 and one copy ofC-AD2-Fab-679. The cancer-targeting antibody component in TF 10 wasderived from hPAM4, a humanized anti-pancreatic cancer mucin MAb thathas been studied in detail as a radiolabeled MAb (e.g., Gold et al.,Clin. Cancer Res. 13: 7380-7387, 2007). The hapten-binding component wasderived from h679, a humanized anti-histaminyl-succinyl-glycine (HSG)MAb. The TF10 bispecific ([hPAM4]₂×h679) antibody was produced using themethod disclosed for production of the (anti CEA)₂×anti HSG bsAb TF2, asdescribed above. The TF10 construct bears two humanized PAM4 Fabs andone humanized 679 Fab.

The two fusion proteins (hPAM4-DDD and h679-AD2) were expressedindependently in stably transfected myeloma cells. The tissue culturesupernatant fluids were combined, resulting in a two-fold molar excessof hPAM4-DDD. The reaction mixture was incubated at room temperature for24 hours under mild reducing conditions using 1 mM reduced glutathione.Following reduction, the DNL reaction was completed by mild oxidationusing 2 mM oxidized glutathione. TF 10 was isolated by affinitychromatography using IMP 291-affigel resin, which binds with highspecificity to the h679 Fab.

A full tissue histology and blood cell binding panel has been examinedfor hPAM4 IgG and for an anti-CEA x anti-HSG bsMAb that is enteringclinical trials. hPAM4 binding was restricted to very weak binding tothe urinary bladder and stomach in 1/3 specimens (no binding was seen invivo), and no binding to normal tissues was attributed to the anti-CEA xanti-HSG bsMAb. Furthermore, in vitro studies against cell lines bearingthe H1 and H2 histamine receptors showed no antagonistic or agonisticactivity with the IMP 288 di-HSG peptide, and animal studies in 2different species showed no pharmacologic activity of the peptiderelated to the histamine component at doses 20,000 times higher thanthat used for imaging. Thus, the HSG-histamine derivative does not havepharmacologic activity.

Example 16 Imaging Studies Using Pretargeting With TF10 BispecificAntibody and ¹¹¹In-Labeled Peptides

The following study demonstrates the feasibility of in vivo imagingusing the pretargeting technique with bispecific antibodiesincorporating hPAM4 and labeled peptides. The TF10 bispecific antibody,comprising two copies of a C-DDD2-Fab-hPAM4 and one copy ofC-AD2-Fab-679, was prepared as described in the preceding Example. Nudemice bearing 0.2 to 0.3 g human pancreatic cancer xenografts wereimaged, using pretargeting with TF10 and ¹¹¹In-IMP-288 peptide. Theresults, shown in FIG. 8A and FIG. 8B, demonstrate how clearlydelineated tumors can be detected in animal models using a bsMAbpretargeting method, with ¹¹¹In-labeled di-HSG peptide, IMP-288. The sixanimals in the top of FIG. 8A and FIG. 8B received 2 different doses ofTF10 (10:1 and 20:1 mole ratio to the moles of peptide given), and thenext day they were given an ¹¹¹In-labeled diHSG peptide (IMP 288). The 3other animals on the bottom of FIG. 8A and FIG. 8B received only the¹¹¹n-IMP-288 (no pretargeting). The images shown in FIG. 8B were taken 3h after the injection of the labeled peptide and show clear localizationof 0.2-0.3 g tumors in the pretargeted animals, with no localization inthe animals given the ¹¹¹In-peptide alone. Tumor uptake averaged 20-25%ID/g with tumor/blood ratios exceeding 2000:1, tumor/liver ratios of170:1, and tumor/kidney ratios of 18/1.

Example 17 Production of Targeting Peptides for Use in Pretargeting and¹⁸F Labeling

In a variety of embodiments, ¹⁸F-labeled proteins or peptides areprepared by a novel technique and used for diagnostic and/or imagingstudies, such as PET imaging. The novel technique for ¹⁸F labelinginvolves preparation of an ¹⁸F-metal complex, preferably an aluminumcomplex, which is chelated to a chelating moiety, such as DOTA, NOTA orNETA or derivatives thereof. Chelating moieties may be attached toproteins, peptides or any other molecule using conjugation techniqueswell known in the art. In certain preferred embodiments, the ¹⁸F-Alcomplex is formed in solution first and then attached to a chelatingmoiety that is already conjugated to a protein or peptide. However, inalternative embodiments the aluminum may be first attached to thechelating moiety and the ¹⁸F added later.

Peptide Synthesis

Peptides were synthesized by solid phase peptide synthesis using theFmoc strategy. Groups were added to the side chains of diamino aminoacids by using Fmoc/Aloc protecting groups to allow differentialdeprotection. The Aloc groups were removed by the method of Dangles et.al. (J. Org. Chem. 1987, 52:4984-4993) except that piperidine was addedin a 1:1 ratio to the acetic acid used. The unsymmetrical tetra-t-butylDTPA was made as described in McBride et al. (US Patent Application Pub.No. US 2005/0002945, the Examples section of which is incorporatedherein by reference).

The tri-t-butyl DOTA, symmetrical tetra-t-butyl DTPA, ITC-benzyl DTPA,p-SCN-Bn-NOTA and TACN were obtained from MACROCYCLICS® (Dallas, Tex.).The DiBocTACN, NODA-GA(tBu)₃ and the NO2AtBu were purchased fromCheMatech (Dijon, France). The Aloc/Fmoc Lysine and Dap(diaminopropionic acid derivatives (also Dpr)) were obtained fromCREOSALUS® (Louisville, Ky.) or BACHEM® (Torrance, Calif.). The SieberAmide resin was obtained from NOVABIOCHEM® (San Diego, Calif.). Theremaining Fmoc amino acids were obtained from CREOSALUS®, BACHEM®,PEPTECH® (Burlington, Mass.), EMD BIOSCIENCES® (San Diego, Calif.), CHEMIMPEX® (Wood Dale, Ill.) or NOVABIOCHEM®. The aluminum chloridehexahydrate was purchased from SIGMA-ALDRICH® (Milwaukee, Wis.). Theremaining solvents and reagents were purchased from FISHER SCIENTIFIC®(Pittsburgh, Pa.) or SIGMA-ALDRICH® (Milwaukee, Wis.). ¹⁸F was suppliedby IBA MOLECULAR® (Somerset, N.J.)

¹⁸F-Labeling of IMP 272

The first peptide that was prepared and ¹⁸F-labeled was IMP 272:

DTPA-Gln-Ala-Lys(HSG)-D-Tyr-Lys(HSG)-NH₂ MH⁺ 1512

IMP 272 was synthesized as described (McBride et al., US PatentApplication Publ. No. 20040241158, the Examples section of which isincorporated herein by reference).

Acetate buffer solution—Acetic acid, 1.509 g was diluted in ˜160 mLwater and the pH was adjusted by the addition of 1 M NaOH then dilutedto 250 mL to make a 0.1 M solution at pH 4.03.

Aluminum acetate buffer solution—A solution of aluminum was prepared bydissolving 0.1028 g of AlCl₃ hexahydrate in 42.6 mL DI water. A 4 mLaliquot of the aluminum solution was mixed with 16 mL of a 0.1 M NaOAcsolution at pH 4 to provide a 2 mM Al stock solution.

IMP 272 acetate buffer solution—Peptide, 0.0011 g, 7.28×10⁻⁷ mol IMP 272was dissolved in 364 μL of the 0.1 M pH 4 acetate buffer solution toobtain a 2 mM stock solution of the peptide.

F-18 Labeling of IMP 272—A 3 μL aliquot of the aluminum stock solutionwas placed in a REACTI-VIAL™ and mixed with 50 μL ¹⁸F (as received) and3 μL of the IMP 272 solution. The solution was heated in a heating blockat 110° C. for 15 min and analyzed by reverse phase HPLC. HPLC analysis(not shown) showed 93% free ¹⁸F and 7% bound to the peptide. Anadditional 10 μL of the IMP 272 solution was added to the reaction andit was heated again and analyzed by reverse phase HPLC (not shown). TheHPLC trace showed 8% ¹⁸F at the void volume and 92% of the activityattached to the peptide. The remainder of the peptide solution wasincubated at room temperature with 150 μL PBS for ˜1 hr and thenexamined by reverse phase HPLC. The HPLC (not shown) showed 58% ¹⁸Funbound and 42% still attached to the peptide. The data indicate that¹⁸F-Al-DTPA complex may be unstable when mixed with phosphate.

The labeled peptide was purified by applying the labeled peptidesolution onto a 1 cc (30 mg) WATERS® HLB column (Part #186001879) andwashing with 300 μL water to remove unbound F-18. The peptide was elutedby washing the column with 2×100 μL 1:1 EtOH/H₂O. The purified peptidewas incubated in water at 25° C. and analyzed by reverse phase HPLC (notshown). The HPLC analysis showed that the ¹⁸F-labeled IMP 272 was notstable in water. After 40 min incubation in water about 17% of the ¹⁸Fwas released from the peptide, while 83% was retained (not shown).

The peptide (16 μL 2 mM IMP 272, 48 μg) was labeled with ¹⁸F andanalyzed for antibody binding by size exclusion HPLC. The size exclusionHPLC showed that the peptide bound hMN-14×679 but did not bind to theirrelevant bispecific antibody hMN-14×734 (not shown).

IMP 272 ¹⁸F Labeling with Other Metals

A ˜3 μL aliquot of the metal stock solution (6×10⁻⁹ mol) was placed in apolypropylene cone vial and mixed with 75 μL ¹⁸F (as received),incubated at room temperature for ˜2 min and then mixed with 20 μL of a2 mM (4×10⁴ mol) IMP 272 solution in 0.1 M NaOAc pH 4 buffer. Thesolution was heated in a heating block at 100° C. for 15 min andanalyzed by reverse phase HPLC. IMP 272 was labeled with indium (24%),gallium (36%), zirconium (15%), lutetium (37%) and yttrium (2%) (notshown). These results demonstrate that the ¹⁸F metal labeling techniqueis not limited to an aluminum ligand, but can also utilize other metalsas well. With different metal ligands, different chelating moieties maybe utilized to optimize binding of an F-18-metal conjugate.

Production and Use of a Serum-Stable ¹⁸F-Labeled Peptide IMP 449

The peptide, IMP 448 D-Ala-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH₂ MH⁺1009 wasmade on Sieber Amide resin by adding the following amino acids to theresin in the order shown: Aloc-D-Lys(Fmoc)-OH, Trt-HSG-OH, the Aloc wascleaved, Fmoc-D-Tyr(But)-OH, Aloc-D-Lys(Fmoc)-OH, Trt-HSG-OH, the Alocwas cleaved, Fmoc-D-Ala-OH with final Fmoc cleavage to make the desiredpeptide. The peptide was then cleaved from the resin and purified byHPLC to produce IMP 448, which was then coupled to ITC-benzyl NOTA. Thepeptide, IMP 448, 0.0757 g (7.5×10⁻⁵ mol) was mixed with 0.0509 g(9.09×10⁻⁵ mol) ITC benzyl NOTA and dissolved in 1 mL water. Potassiumcarbonate anhydrous (0.2171 g) was then slowly added to the stirredpeptide/NOTA solution. The reaction solution was pH 10.6 after theaddition of all the carbonate. The reaction was allowed to stir at roomtemperature overnight. The reaction was carefully quenched with 1 M HClafter 14 hr and purified by HPLC to obtain 48 mg of IMP 449.

¹⁸F Labeling of IMP 449

The peptide IMP 449 (0.002 g, 1.37×10⁻⁶ mol) was dissolved in 686 μL (2mM peptide solution) 0.1 M NaOAc pH 4.02. Three microliters of a 2 mMsolution of Al in a pH 4 acetate buffer was mixed with 15 μL, 1.3 mCi of¹⁸F. The solution was then mixed with 20 μL of the 2 mM IMP 449 solutionand heated at 105° C. for 15 min. Reverse Phase HPLC analysis showed 35%(t_(R)˜10 min) of the activity was attached to the peptide and 65% ofthe activity was eluted at the void volume of the column (3.1 min, notshown) indicating that the majority of activity was not associated withthe peptide. The crude labeled mixture (5 μL) was mixed with pooledhuman serum and incubated at 37° C. An aliquot was removed after 15 minand analyzed by HPLC. The HPLC showed 9.8% of the activity was stillattached to the peptide (down from 35%). Another aliquot was removedafter 1 hr and analyzed by HPLC. The HPLC showed 7.6% of the activitywas still attached to the peptide (down from 35%), which was essentiallythe same as the 15 min trace (data not shown).

High Dose ¹⁸F Labeling

Further studies with purified IMP 449 demonstrated that the ¹⁸F-labeledpeptide was highly stable (91%, not shown) in human serum at 37° C. forat least one hour and was partially stable (76%, not shown) in humanserum at 37° C. for at least four hours. Additional studies wereperformed in which the IMP 449 was prepared in the presence of ascorbicacid as a stabilizing agent. In those studies (not shown), themetal-¹⁸F-peptide complex showed no detectable decomposition in serumafter 4 hr at 37° C. The mouse urine 30 min after injection of¹⁸F-labeled peptide was found to contain ¹⁸F bound to the peptide (notshown). These results demonstrate that the ¹⁸F-labeled peptidesdisclosed herein exhibit sufficient stability under approximated in vivoconditions to be used for ¹⁸F imaging studies.

For studies in the absence of ascorbic acid, ¹⁸F˜21 mCi in ˜400 μL ofwater was mixed with 9 μL of 2 mM AlCl₃ in 0.1 M pH 4 NaOAc. Thepeptide, IMP 449, 60 μL (0.01 M, 6×10⁻⁷ mol in 0.5 NaOH pH 4.13) wasadded and the solution was heated to 110° C. for 15 min. The crudelabeled peptide was then purified by placing the reaction solution inthe barrel of a 1 cc WATERS® HLB column and eluting with water to removeunbound ¹⁸F followed by 1:1 EtOH/H₂O to elute the ¹⁸F-labeled peptide.The crude reaction solution was pulled through the column into a wastevial and the column was washed with 3×1 mL fractions of water (18.97mCi). The HLB column was then placed on a new vial and eluted with 2×200μL 1:1 EtOH/H₂O to collect the labeled peptide (1.83 mCi). The columnretained 0.1 mCi of activity after all of the elutions were complete. Analiquot of the purified ¹⁸F-labeled peptide (20 μL) was mixed with 200μL of pooled human serum and heated at 37° C. Aliquots were analyzed byreverse phase HPLC. The results showed the relative stability of¹⁸F-labeled purified IMP 449 at 37° C. at time zero, one hour (91%labeled peptide), two hours (77% labeled peptide) and four hours (76%labeled peptide) of incubation in human serum (not shown). It was alsoobserved that ¹⁸F-labeled IMP 449 was stable in TFA solution, which isoccasionally used during reverse phase HPLC chromatography. Thereappears to be a general correlation between stability in TFA andstability in human serum observed for the exemplary ¹⁸F-labeledmolecules described herein. These results demonstrate that ¹⁸F-labeledpeptide, produced according to the methods disclosed herein, showssufficient stability in human serum to be successfully used for in vivolabeling and imaging studies, for example using PET scanning to detectlabeled cells or tissues. Finally, since IMP 449 peptide contains athiourea linkage, which is sensitive to radiolysis, several products areobserved by RP-HPLC. However, when ascorbic acid is added to thereaction mixture, the side products generated were markedly reduced.

Example 18 In Vivo Studies with Pretargeting TF10 DNL Construct and¹⁸F-Labeled Peptide

¹⁸F-labeled IMP 449 was prepared as follows. The ¹⁸F, 54.7 mCi in 0.5 mLwas mixed with 3 μL 2 mM Al in 0.1 M NaOAc pH 4 buffer. After 3 min 10μL of 0.05 M IMP 449 in 0.5 M pH 4 NaOAc buffer was added and thereaction was heated in a 96° C. heating block for 15 min. The contentsof the reaction were removed with a syringe. The crude labeled peptidewas then purified by HPLC on a C₁₈ column. The flow rate was 3 mL/min.Buffer A was 0.1% TFA in water and Buffer B was 90% acetonitrile inwater with 0.1% TFA. The gradient went from 100% A to 75/25 A:B over 15min. There was about 1 min difference in retention time (t_(R)) betweenthe labeled peptide, which eluted first and the unlabeled peptide. TheHPLC eluent was collected in 0.5 min (mL) fractions. The labeled peptidehad a t_(R) between 6 to 9 min depending on the column used. The HPLCpurified peptide sample was further processed by diluting the fractionsof interest two fold in water and placing the solution in the barrel ofa 1 cc WATERS® HLB column. The cartridge was eluted with 3×1 mL water toremove acetonitrile and TFA followed by 400 μL 1:1 EtOH/H₂O to elute the¹⁸F-labeled peptide. The purified [Al¹⁸F] IMP 449 eluted as a singlepeak on an analytical HPLC C₁₈ column (not shown).

TACONIC® nude mice bearing the four slow-growing sc CaPan1 xenograftswere used for in vivo studies. Three of the mice were injected with TF10(162 μg) followed with [Al¹⁸F] IMP 449 18 h later. TF10 is a humanizedbispecific antibody of use for tumor imaging studies, with divalentbinding to the PAM-4 defined tumor antigen and monovalent binding to HSG(see, e.g., Gold et al., 2007, J. Clin. Oncol. 25(18S):4564). One mousewas injected with peptide alone. All of the mice were necropsied at 1 hpost peptide injection. Tissues were counted immediately. Animal #2showed high counts in the femur. The femur was transferred into a newvial and was recounted along with the old empty vial. Recountingindicated that the counts were on the tissue. This femur was broken andhad a large piece of muscle attached to it. Comparison of meandistributions showed substantially higher levels of ¹⁸F-labeled peptidelocalized in the tumor than in any normal tissues in the presence oftumor-targeting bispecific antibody.

Tissue uptake was similar in animals given the [Al¹⁸F] IMP 449 alone orin a pretargeting setting (Table 9). Uptake in the human pancreaticcancer xenograft, CaPan1, at 1 h was increased 5-fold in the pretargetedanimals as compared to the peptide alone (4.6±0.9% ID/g vs. 0.89% ID/g).Exceptional tumor/nontumor ratios were achieved at this time (e.g.,tumor/blood and liver ratios were 23.4±2.0 and 23.5±2.8, respectively).

TABLE 9 Tissue uptake at 1 h post peptide injection, mean and theindividual animals: [Al¹⁸F] IMP 449 TF10 (162 μg) -→18 h → [Al¹⁸F] IMP449 (10:1) alone Tissue n Mean SD Animal 1 Animal 2 Animal 3 Animal 1Tumor 3 4.591 0.854 4.330 5.546 3.898 0.893 (mass) (0.675 g) (0.306 g)(0.353 g) (0.721 g) Liver 3 0.197 0.041 0.163 0.242 0.186 0.253 Spleen 30.202 0.022 0.180 0.224 0.200 0.226 Kidney 3 5.624 0.531 5.513 6.2025.158 5.744 Lung 3 0.421 0.197 0.352 0.643 0.268 0.474 Blood 3 0.1960.028 0.204 0.219 0.165 0.360 Stomach 3 0.123 0.046 0.080 0.172 0.1180.329 Small Int. 3 0.248 0.042 0.218 0.295 0.230 0.392 Large Int. 30.141 0.094 0.065 0.247 0.112 0.113 Pancreas 3 0.185 0.078 0.259 0.1940.103 0.174 Spine 3 0.394 0.427 0.140 0.888 0.155 0.239 Femur 3 3.8994.098 2.577 8.494 0.625 0.237 Brain 3 0.064 0.041 0.020 0.072 0.1000.075 Muscle 3 0.696 0.761 0.077 1.545 0.465 0.162

The results demonstrate that ¹⁸F labeled peptide used in conjunctionwith a PAM4 containing antibody construct, such as the TF10 DNLconstruct, provide suitable targeting of the ¹⁸F label to perform invivo imaging, such as PET imaging analysis.

Example 19 Further Imaging Studies with TF10

Summary

Preclinical and clinical studies have demonstrated the application ofradiolabeled mAb-PAM4 for nuclear imaging and radioimmunotherapy ofpancreatic carcinoma. We have examined herein the ability of the novelPAM4-based, bispecific monoclonal antibody (mAb) construct, TF10, topretarget a radiolabeled peptide for improved imaging and therapy. TF10is a humanized, bispecific mAb, divalent for mAb-PAM4 and monovalent formAb-679, reactive against the histamine-succinyl-glycine hapten.Biodistribution studies and nuclear imaging of the radiolabeled TF10and/or TF10-pretargeted hapten-peptide (IMP-288) were conducted in nudemice bearing CaPan1 human pancreatic cancer xenografts. ¹²⁵I-TF10cleared rapidly from the blood, with levels decreasing to <1% injecteddose per gram (ID/g) by 16 hours. Tumor uptake was 3.47±0.66% ID/g atthis time point with no accumulation in any normal tissue. To show theutility of the pretargeting approach, ¹¹¹In-IMP-288 was administered 16hours after TF10. At 3 hours postadministration of radiolabeled peptide,imaging showed intense uptake within the tumors and no evidence ofaccretion in any normal tissue (Example 16). No targeting was observedin animals given only the ¹¹¹In-peptide (Example 16). Tumor uptake ofthe TF10-pretargeted ¹¹¹In-IMP-288 was 24.3±1.7% ID/g, whereas for¹¹¹In-IMP-288 alone it was only 0.12±0.002% ID/g at 16 hours.Tumor/blood ratios were significantly greater for the pretargeting group(˜1,000:1 at 3 hours) compared with ¹¹¹In-PAM4-IgG (˜5:1 at 24 hours;P<0.0003). Radiation dose estimates suggested that TF 10/⁹⁰Y-peptidepretargeting would provide a greater antitumor effect than ⁹⁰Y-PAM4-IgG.Thus, the results support that TF 10 pretargeting may provide improvedimaging for early detection, diagnosis, and treatment of pancreaticcancer as compared with directly radiolabeled PAM4-IgG. (Gold et al.,Cancer Res 2008, 68(12):4819-26)

We have identified a unique biomarker present on mucin expressed by >85%of invasive pancreatic adenocarcinomas, including early stage I diseaseand the precursor lesions, pancreatic intraepithelial neoplasia andintraductal papillary mucinous neoplasia (Gold et al., Clin Cancer Res2007, 13:7380-87). The specific epitope, as detected by mAb-PAM4 (Goldet al., Int J Cancer 1994, 57:204-10), is absent from normal andinflamed pancreatic tissues, as well as most other malignant tissues.Thus, detection of the epitope provides a high diagnostic likelihood forthe presence of pancreatic neoplasia. Early clinical studies using ¹³¹I-and ^(99m)Tc-labeled, murine PAM4 IgG or Fab′, respectively, showedspecific targeting in 8 of 10 patients with invasive pancreaticadenocarcinoma (Mariani et al., Cancer Res 1995, 55:5911s-15s; Gold etal., Crit Rev Oncol Hematol 2001, 39:147-54). Of the two negativepatients, one had a poorly differentiated pancreatic carcinoma that didnot express the PAM4-epitope, whereas the other patient was later foundto have pancreatitis rather than a malignant lesion.

Accordingly, the high specificity of PAM4 for pancreatic cancer is ofuse for the detection and diagnosis of early disease. In addition toimproved detection, ⁹⁰Y-PAM4 IgG was found to be effective in treatinglarge human pancreatic cancer xenografts in nude mice (Cardillo et al.,Clin Cancer Res 2001, 7:3186-92), and when combined with gemcitabine,further improvements in therapeutic response were observed (Gold et al.,Clin Cancer Res 2004, 10:3552-61; Gold et al., Int J Cancer 2004,109:618-26). A Phase I therapy trial in patients who failed gemcitabinetreatment was recently completed, finding the maximum tolerated dose of⁹⁰Y-humanized PAM4 IgG to be 20 mCi/m² (Gulec et al., Proc Amer Soc ClinOne, 43rd Annual Meeting, J Clin Oncol 2007, 25(18S):636s). Although allpatients showed disease progression at or after week 8, initialshrinkage of tumor was observed in several cases. Clinical studies arenow underway to evaluate a fractionated dosing regimen of ⁹⁰Y-hPAM4 IgGin combination with a radiosensitizing dose of gemcitabine.

We report herein the development of a novel recombinant, humanizedbispecific monoclonal antibody (mAb), TF10, based on the targetingspecificity of PAM4 to pancreatic cancer. This construct also binds tothe unique synthetic hapten, histamine-succinyl-glycine (HSG), which hasbeen incorporated in a number of small peptides that can be radiolabeledwith a wide range of radionuclides suitable for single-photon emissioncomputed tomography (SPECT) and positron emission tomography (PET)imaging, as well as for therapeutic purposes (Karacay et al., ClinCancer Res 2005, 11:7879-85; Sharkey et al., Leukemia 2005, 19:1064-9;Rossi et al., Proc Natl Acad Sci USA 2006, 103:6841-6; McBride et al., JNucl Med 2006, 47:1678-88). These studies illustrate the potential ofthis new construct to target pancreatic adenocarcinoma for imaging ortherapeutic applications.

Methods and Materials

The TF2 and TF10 bispecific DNL constructs and the IMP 288 targetingpeptide were prepared as described above. Sodium iodide (¹²⁵I) andindium chloride (¹¹¹In) were obtained from PERKIN-ELMER®. TF10 wasroutinely labeled with ¹²⁵I by the iodogen method, with purification byuse of size-exclusion spin columns. Radiolabeling of DOTA-peptide andDOTA-PAM4-IgG with ¹¹¹InCl was done as previously described (Rossi etal., Proc Natl Acad Sci U S A 2006, 103:6841-6; McBride et al., J NuclMed 2006, 47:1678-88). Purity of the radiolabeled products was examinedby size-exclusion high-performance liquid chromatography with the amountof free, unbound isotope determined by instant TLC.

For TF10 distribution studies, female athymic nude mice ˜20 g (TACONIC®Farms), bearing s.c. CaPan1 human pancreatic cancer xenografts, wereinjected with ¹²⁵I-TF10 (10 μCi; 2.50×10⁻¹⁰ mol). At various timepoints, groups of mice (n=5) were necropsied, with tumor and nontumortissues removed and counted in a gamma counter to determine thepercentage of injected dose per gram of tissue (% ID/g), with thesevalues used to calculate blood clearance rates and tumor/nontumorratios.

For pretargeting biodistribution studies, a bispecific mAb/radiolabeledpeptide molar ratio of 10:1 was used. For example, a group of athymicnude mice bearing s.c. CaPan1 human pancreatic cancer xenografts wasadministered TF10 (80 μg, 5.07×10⁻¹⁰ mol), whereas a second group wasleft untreated. At 16 h postinjection of TF10, ¹¹¹In-IMP-288hapten-peptide (30 μCi, 5.07×10⁻¹¹ mol) was administered. Mice werenecropsied at several time points, with tumor and nontumor tissuesremoved and counted in a gamma counter to determine the % ID/g.Tumor/nontumor ratios were calculated from these data. In a separatestudy, groups of mice were given ¹¹¹In-DOTA-PAM4-IgG (20 μC, 50 μg,3.13×10⁻¹⁰ mol) for the purpose of comparing biodistribution, nuclearimaging, and potential therapeutic activity. Radiation dose estimateswere calculated from the time-activity curves with the assumption of noactivity at zero time. Student's t test was used to assess significantdifferences.

To perform nuclear immuoscintigraphy, at 3 h postinjection ofradiolabeled peptide or 24 h postinjection of radiolabeled hPAM4-IgG,tumor-bearing mice were imaged with a dual-head Solus gamma camerafitted with medium energy collimator for ¹¹¹In (ADAC Laboratories). Micewere imaged for a total of 100,000 cpm or 10 min, whichever came first.

Results

In Vitro Characterization of the Bispecific mAb TF10.

The binding of TF10 to the target mucin antigen was analyzed by ELISA(FIG. 9). The results showed nearly identical binding curves for thedivalent TF10, PAM4-IgG, and PAM4-F(ab′)₂ (half-maximal binding wascalculated as 1.42±0.10, 1.31±0.12, and 1.83±0.16 nmol/L, respectively;P>0.05 for all), whereas the monovalent bsPAM4 chemical conjugate(PAM4-Fab′×anti-DTPA-Fab′) had a significantly lower avidity(half-maximal binding, 30.61±2.05 nmol/L; P=0.0379, compared with TF10),suggesting that TF10 binds in a divalent manner. The immunoreactivefraction of ¹²⁵I-TF10 bound to the mucin was 87%, with 9% found asunbound TF10 and 3% as free iodide (not shown). Ninety percent of the¹¹¹In-IMP-288 bound to TF10 (not shown). Of the total ¹¹¹In-IMP-288bound to TF10, 92% eluted at higher molecular weight when excess mucin(200 μg) was added, with only 3% eluting with the non-mucin-reactive TF10 fraction. An additional 5% of the radiolabeled peptide eluted in thefree peptide volume. None of the radiolabeled peptide bound to the mucinantigen in the absence of TF10 (not shown).

Biodistribution of ¹²⁵I-TF10 in CaPan1 Tumor-Bearing Nude Mice.

TF10 showed a rapid clearance from the blood, starting with 21.03±1.93%ID/g at 1 hour and decreasing to just 0.13±0.02% ID/g at 16 hours. Thebiological half-life was calculated to be 2.19 hours [95% confidenceinterval (95% CI), 2.11-2.27 hours]. Tissue uptake revealed enhancedactivity in the liver, spleen, and kidneys at 1 hour, which cleared justas quickly by 16 hours [T_(1/2)=2.09 hours (95% CI, 2.08-2.10), 2.84hours (95% CI, 2.49-3.29), and 2.44 hours (95% CI, 2.28-2.63) for liver,spleen, and kidney, respectively]. Activity in the stomach most likelyreflects the accretion and excretion of radioiodine, suggesting that theradioiodinated TF 10 was actively catabolized, presumably in the liverand spleen, thereby explaining its rapid clearance from the blood.Nevertheless, by 16 hours, the concentration of radioiodine within thestomach was below 1% ID/g. A group of five non-tumor-bearing nude micegiven ¹²⁵I-TF10 and necropsied at 16 hours showed similar tissuedistribution, suggesting that the tumor had not affected the bispecificmAb distribution and clearance from normal tissues (data not shown). Ofcourse, it is possible that differences occurred before the initial timepoint examined. Tumor uptake of TF10 peaked at 6 hours postinjection(7.16±1.10% ID/g) and had decreased to half maximum binding (3.47±0.66%ID/g) at 16 hours. Tumor uptake again decreased nearly 2-fold over thenext 32 hours, but then was stable over the following 24 hours.

Biodistribution of TF10-Pretargeted, ¹¹¹In-Labeled Peptide.

Although maximum tumor uptake of TF10 occurred at 6 hours, previousexperience indicated that the radiolabeled peptide would need to begiven at a time point when blood levels of TF 10 had cleared to <1% ID/g(i.e., 16 hours). Higher levels of TF10 in the blood would lead tounacceptably high binding of the radiolabeled peptide within the blood(i.e., low tumor/blood ratios), whereas administering the peptide at alater time would mean the concentration of TF10 in the tumor would bedecreased with consequently reduced concentration of radiolabeledpeptide within the tumor. Thus, an initial pretargeting study was doneusing a 16-hour interval. With the amount of the ¹¹¹In-IMP-288 heldconstant (30 μCi, 5.07×10⁻¹¹ mol), increasing amounts of TF10 were givenso that the administered dose of TF10 and IMP-288 expressed as moleratio varied from 5:1 to 20:1 (Table 10).

TABLE 10 Biodistribution of ¹¹¹In-IMP-288 alone (no TF10) or pretargetedwith varying amounts of TF10 % ID/g at 3 h (mean ± SD) 5:1 10:1 20:1 NoTF10 Tumor 19.0 ± 3.49 24.3 ± 1.71 28.6 ± 0.73 0.12 ± 0.00 Liver 0.09 ±0.01 0.21 ± 0.12 0.17 ± 0.01 0.07 ± 0.00 Spleen 0.12 ± 0.04 0.16 ± 0.070.26 ± 0.10 0.04 ± 0.01 Kidneys 1.59 ± 0.11 1.72 ± 0.24 1.53 ± 0.14 1.71± 0.22 Lungs 0.19 ± 0.06 0.26 ± 0.00 0.29 ± 0.04 0.03 ± 0.00 Blood 0.01± 0.00 0.01 ± 0.01 0.01 ± 0.00 0.00 ± 0.00 Stomach 0.03 ± 0.02 0.02 ±0.02 0.01 ± 0.00 0.02 ± 0.01 Small intestine 0.12 ± 0.08 0.08 ± 0.030.04 ± 0.01 0.06 ± 0.02 Large intestine 0.23 ± 0.10 0.39 ± 0.08 0.25 ±0.08 0.33 ± 0.02 Pancreas 0.02 ± 0.00 0.02 ± 0.01 0.02 ± 0.00 0.02 ±0.00 Tumor weight 0.12 ± 0.03 0.32 ± 0.09 0.27 ± 0.01 0.35 ± 0.03 (g)

At 3 hours the amount of ¹¹¹In-IMP-288 in the blood was barelydetectable (0.01%). Tumor uptake increased from 19.0±3.49% ID/g to28.55±0.73% ID/g as the amount of bispecific mAb administered wasincreased 4-fold (statistically significant differences were observedfor comparison of each TF10/peptide ratio, one group to another; P<0.03or better), but without any appreciable increase in normal tissueuptake. Tumor uptake in the animals given TF10 was >100-fold higher thanwhen ¹¹¹In-IMP-288 was given alone. Comparison of ¹¹¹In activity in thenormal tissues of the animals that either received or did not receiveprior administration of TF10 indicated similar absolute values, which inmost instances were not significantly different. This suggests that thebispecific mAb had cleared sufficiently from all normal tissues by 16hours to avoid appreciable peptide uptake in these tissues. Tumor/bloodratios were >2,000:1, with other tissue ratios exceeding 100:1. Eventumor/kidney ratios exceeded 10:1. The highest tumor uptake ofradioisotope with minimal targeting to nontumor tissues resulted fromthe 20:1 ratio; however, either of the TF10/peptide ratios could be usedto achieve exceptional targeting to tumor, both in terms of signalintensity and contrast ratios. The 10:1 ratio was chosen for furtherstudy because the absolute difference in tumor uptake of radiolabeledpeptide was not substantially different between the 10:1 (24.3±1.71%ID/g) and 20:1 (28.6±0.73% ID/g) ratios.

Images of the animals given TF10-pretargeted ¹¹¹In-IMP-288 at abispecific mAb/peptide ratio of 10:1, or the ¹¹¹In-IMP-288 peptidealone, are shown in FIG. 10A, FIG. 10B and FIG. 10C. The majority ofthese tumors were ≦0.5 cm in diameter, weighing ˜0.25 g. The images showhighly intense uptake in the tumor of the TF10-pretargeted animals (FIG.10A). The intensity of the image background for the TF10-pretargetedanimals was increased to match the intensity of the image taken of theanimals given the ¹¹¹In-IMP-288 alone (FIG. 10B). However, when theimages were optimized for the TF10-pretargeted mice, the signalintensity and contrast were so high that no additional activity wasobserved in the body. No tumor localization was seen in the animalsgiven the ¹¹¹In-IMP-288alone, even when image intensity was enhanced(FIG. 10C).

An additional experiment was done to assess the kinetics of targeting¹¹¹In-hPAM4 whole-IgG compared with that of theTF10-pretargeted¹¹¹In-IMP-288 peptide. Tumor uptake of the ¹¹¹In-peptidewas highest at the initial time point examined, 3 hours (15.99±4.11%ID/g), whereas the blood concentration of radiolabeled peptide was only0.02±0.01% ID/g, providing a mean tumor/blood ratio of 946.3±383.0. Overtime, radiolabeled peptide cleared from the tumor with a biologicalhalf-life of 76.04 hours. Among nontumor tissues, uptake was highest inthe kidneys, averaging 1.89±0.42% ID/g at 3 hours with a steady decreaseover time (biological half-life, 33.6 hours). Liver uptake started at0.15±0.06% ID/g and remained essentially unchanged over time. Incontrast to the TF10-pretargeted¹¹¹In-IMP-288, the ¹¹¹In-hPAM4-IgG had aslower clearance from the blood, albeit there was a substantialclearance within the first 24 hours, decreasing from 30.1% ID/g at 3hours to just 11.5±1.7% ID/g at 24 hours. Variable elevated uptake inthe spleen suggested that the antibody was likely being removed from theblood by targeting of secreted mucin that had become entrapped withinthe spleen. Tumor uptake peaked at 48 hours with 80.4±6.1% ID/g, andremained at an elevated level over the duration of the monitoringperiod. The high tumor uptake, paired with a more rapid than expectedblood clearance for an IgG, produced tumor/blood ratios of 5.2±1.0within 24 hours. FIG. 10C shows the images of the animals at 24 hourspostadministration of ¹¹¹In-PAM4-IgG, illustrating that tumors could bevisualized at this early time, but there was still considerable activitywithin the abdomen. Tumor/nontumor ratios were mostly higher forTF10-pretargeted ¹¹¹In-labeled hapten-peptide as compared with¹¹¹In-hPAM4-IgG, except for the kidneys, where tumor/kidney ratios withthe ¹¹¹In-IMP-288 and ¹¹¹In-hPAM4-IgG were similar at later times.However, tumor/kidney ratios for the TF10-pretargeted ¹¹¹In-IMP-288 werehigh enough (e.g., ˜7:1) at 3 hours to easily discern tumor from normaltissue.

FIG. 11A to FIG. 11D illustrates the potential therapeutic capability ofthe direct and pretargeted methods to deliver radionuclide (⁹⁰Y).Although the concentration (% ID/g) of radioisotope within the tumorseems to be much greater when delivered by PAM4-IgG than by pretargetedTF10 at their respective maximum tolerated dose (0.15 mCi for ⁹⁰Y-hPAM4and 0.9 mCi for TF10-pretargeted ⁹⁰Y-IMP-288) (FIG. 11A), the radiationdose to tumor would be similar (10,080 and 9,229 cGy for ⁹⁰Y-PAM4-IgGand TF10-pretargeted ⁹⁰Y-IMP-288, respectively) (FIG. 11C). Theadvantage for the pretargeting method would be the exceptionally lowactivity in blood (9 cGy), almost 200-fold less than with the ⁹⁰Y-hPAM4IgG (1,623 cGy) (FIG. 11C). It is also important to note that theradiation dose to liver, as well as other nontumor organs, would be muchlower with the TF10-pretargeted ⁹⁰Y-IMP-288 (FIG. 11B, FIG. 11D). Theexception would be the kidneys, where the radiation dose would besimilar for both protocols at their respective maximum dose (612 and 784cGy for ⁹⁰Y-PAM4-IgG and TF10-⁹⁰Y-IMP-288, respectively) (FIG. 11B, FIG.11D). The data suggest that for ⁹⁰Y-PAM4-IgG, as with most otherradiolabeled whole-IgG mAbs, the dose-limiting toxicity would behematologic; however, for the TF10 pretargeting protocol, thedose-limiting toxicity would be the kidneys.

Discussion

Current diagnostic modalities such as ultrasound, computerizedtomography (CT), and magnetic resonance imaging (MRI) technologies,which provide anatomic images, along with PET imaging of the metabolicenvironment, have routinely been found to provide high sensitivity inthe detection of pancreatic masses. However, these data are, for themost part, based on detection of lesions>2 cm in a population that isalready presenting clinical symptoms. At this time in the progression ofthe pancreatic carcinoma, the prognosis is rather dismal. To improvepatient outcomes, detection of small, early pancreatic neoplasms in theasymptomatic patient is necessary.

Imaging with a mAb-targeted approach, such as is described herein withmAb-PAM4, may provide for the diagnosis of these small, early cancers.Of prime importance is the specificity of the mAb. We have presentedconsiderable data, including immunohistochemical studies of tissuespecimens (Gold et al., Clin Cancer Res 2007; 13:7380-7; Gold et al.,Int J Cancer 1994; 57:204-10) and immunoassay of patient sera (Gold etal., J Clin Oncol 2006; 24:252-8), to show that mAb-PAM4 is highlyreactive with a biomarker, the presence of which provides highdiagnostic likelihood of pancreatic neoplasia. Furthermore, wedetermined that PAM4, although not reactive with normal adult pancreatictissues nor active pancreatitis, is reactive with the earliest stages ofneoplastic progression within the pancreas (pancreatic intraepithelialneoplasia 1 and intraductal papillary mucinous neoplasia) and that thebiomarker remains at high levels of expression throughout theprogression to invasive pancreatic adenocarcinoma (Gold et al., ClinCancer Res 2007; 13:7380-7). Preclinical studies with athymic nude micebearing human pancreatic tumor xenografts have shown specific targetingof radiolabeled murine, chimeric, and humanized versions of PAM4.

In the current studies, we have examined a next-generation, recombinant,bispecific PAM4-based construct, TF10, which is divalent for the PAM4arm and monovalent for the anti-HSG hapten arm. There are severalimportant characteristics of this pretargeting system's constructs,named dock-and-lock, including its general applicability and ease ofsynthesis. However, for the present consideration, the major differencesfrom the previously reported chemical construct are the valency, whichprovides improved binding to tumor antigen, and, importantly, itspharmacokinetics. TF10 clearance from nontumor tissues is much morerapid than was observed for the chemical conjugate. Time for bloodlevels of the bispecific constructs to reach less than 1% ID/g was 40hours postinjection for the chemical construct versus 16 hours for TF10.A more rapid clearance of the pretargeting agent has provided a vastimprovement of the tumor/blood ratio, while maintaining high signalstrength at the tumor site (% ID/g).

In addition to providing a means for early detection and diagnosis, theresults support the use of the TF10 pretargeting system for cancertherapy. Consideration of the effective radiation dose to tumor andnontumor tissues favors the pretargeting method over directlyradiolabeled PAM4-IgG. The dose estimates suggest that the two deliverysystems have different dose-limiting toxicities: myelotoxicity for thedirectly radiolabeled PAM4 versus the kidney for the TF10 pretargetingsystem. This is of significance for the future clinical development ofradiolabeled PAM4 as a therapeutic agent. Gemcitabine, the frontlinedrug of choice for pancreatic cancer, can provide significantradiosensitization of tumor cells. In previous studies, we showed thatcombinations of gemcitabine and directly radiolabeled PAM4-IgG providedsynergistic antitumor effects compared with either arm alone (Gold etal., Clin Cancer Res 2004, 10:3552-61; Gold et al., Int J Cancer 2004,109:618-26). The dose-limiting factor with this combination wasoverlapping hematologic toxicity. However, because the dose-limitingorgan for TF10 pretargeting seems to be the kidney rather thanhematologic tissues, combinations with gemcitabine should be less toxic,thus allowing increased administration of radioisotope with consequentlygreater antitumor efficacy.

The superior imaging achieved with TF 10 pretargeting in preclinicalmodels, as compared with directly radiolabeled DOTA-PAM4-IgG, provides acompelling rationale to proceed to clinical trials with this imagingsystem. The specificity of the tumor-targeting mAb for pancreaticneoplasms, coupled with the bispecific antibody platform technologyproviding the ability to conjugate various imaging compounds to theHSG-hapten-peptide for SPECT (¹¹¹In), PET (⁶⁸Ga), ultrasound (Au), orother contrast agents, or for that matter ⁹⁰Y or other radionuclides fortherapy, provides high potential to improve overall patient outcomes(Goldenberg et al., J Nucl Med 2008, 49:158-63). In particular, webelieve that a TF10-based ImmunoPET procedure will have major clinicalvalue to screen individuals at high-risk for development of pancreaticcancer (e.g., genetic predisposition, chronic pancreatitis, smokers,etc.), as well as a means for follow-up of patients with suspiciousabdominal images from conventional technologies and/or with indicationsdue to the presence of specific biomarker(s) or abnormal biochemicalfindings. When used as part of an ongoing medical plan for followingthese patients, early detection of pancreatic cancer may be achieved.Finally, in combination with gemcitabine, TF10 pretargeting may providea better opportunity for control of tumor growth than directlyradiolabeled PAM4-IgG.

Example 20 Therapy of Pancreatic Cancer Xenografts with Gemcitabine and⁹⁰Y-Labeled Peptide Pretargeted Using TF10

Summary

⁹⁰Y-hPAM4 IgG is currently being examined in Phase I/II trials incombination with gemcitabine in patients with Stage III/IV pancreaticcancer. We disclose a new approach for pretargeting radionuclides thatis able to deliver a similar amount of radioactivity to pancreaticcancer xenografts, but with less hematological toxicity, which would bemore amenable for combination with gemcitabine. Nude mice bearing ˜0.4cm³ sc CaPan1 human pancreatic cancer were administered a recombinantbsMAb, TF 10, followed 1 day later with a ⁹⁰Y-labeled hapten-peptide(IMP-288). Various doses and schedules of gemcitabine were added to thistreatment, and tumor progression monitored up to 28 weeks. 0.7 mCiPT-RAIT alone produce only a transient 60% loss in blood counts, andanimals given 0.9 mCi of PT-RAIT alone and 0.7 mCi PT-RAIT+6 mggemcitabine (human equivalent ˜1000 mg/m²) had no histological evidenceof renal toxicity after 9 months. A single dose of 0.25 or 0.5 mCiPT-RAIT alone can completely ablate 20% and 80% of the tumors,respectively. Monthly fractionated PT-RAIT (0.25 mCi/dose given at thestart of each gemcitabine cycle) added to a standard gemcitabine regimen(6 mg wkly×3; 1 wk off; repeat 3 times) significantly increased themedian time for tumors to reach 3.0 cm³ over PT-RAIT alone. Othertreatment plans examining non-cytotoxic radiosensitizing dose regimensof gemcitabine added to PT-RAIT also showed significant improvements intreatment response over PT-RAIT alone. The results show that PT-RAIT isa promising new approach for treating pancreatic cancer. Current dataindicate combining PT-RAIT with gemcitabine will enhance therapeuticresponses.

Methods

TF10 bispecific antibody was prepared as described above. Forpretargeting, TF10 was given to nude mice bearing the human pancreaticadenocarcinoma cell line, CaPan1. After allowing sufficient time forTF10 to clear from the blood (16 h), the radiolabeled divalentHSG-peptide was administered. The small molecular weight HSG-peptide(˜4.4 kD) clears within minutes from the blood, entering theextravascular space where it can bind to anti-HSG arm of the pretargetedTF10 bsMAb. Within a few hours, >80% of the radiolabeled HSG-peptide isexcreted in the urine, leaving the tumor localized peptide and a traceamount in the normal tissues.

Results

FIG. 12 illustrates the therapeutic activity derived from a singletreatment of established (˜0.4 cm³) CaPan1 tumors with 0.15 mCi of⁹⁰Y-hPAM4 IgG, or 0.25 or 0.50 mCi of TF10-pretargeted ⁹⁰Y-IMP-288.Similar anti-tumor activity was observed for the 0.5-mCi pretargeteddose vs. 0.15-mCi dose of the directly radiolabeled IgG, buthematological toxicity was severe at this level of the direct conjugate(not shown), while the pretargeted dose was only moderately toxic (notshown). Indeed, the MTD for pretargeting using 90Y-IMP-288 is at least0.9 mCi in nude mice.

FIG. 13 shows that the combination of gemcitabine and PT-RAIT has asynergistic effect on anti-tumor therapy. Human equivalent doses of 1000mg/m² (6 mg) of gemcitabine (GEM) were given intraperitoneally to miceweekly for 3 weeks, then after resting for 1 week, this regimen wasrepeated 2 twice. PT-RAIT (0.25 mCi of TF10-pretargeted ⁹⁰Y-IMP-288) wasgiven 1 day after the first GEM dose in each of the 3 cycles oftreatment. Gem alone had no significant impact on tumor progression(survival based on time to progress to 3.0 cm³). PT-RAIT alone improvedsurvival compared to untreated animals, but the combined GEM withPT-RAIT regimen increased the median survival by nearly 10 weeks.Because hematological toxicity is NOT dose-limiting for PT-RAIT, but itis one of the limitations for gemcitabine therapy, these studies suggestthat PT-RAIT could be added to a standard GEM therapy with the potentialfor enhanced responses. The significant synergistic effect ofgemcitabine plus PT-RAIT was surprising and unexpected.

A further study examined the effect of the timing of administration onthe potentiation of anti-tumor effect of gemcitabine plus PT-RAIT. Asingle 6-mg dose of GEM was given one day before or 1 day after 0.25 mCiof TF10-pretargeted ⁹⁰Y-IMP-288 (not shown). This study confirmed whatis already well known with GEM, i.e., radiosensitization is best givenin advance of the radiation. Percent survival of treated mice showedlittle difference in survival time between PT-RAIT alone and PT-RAITwith gemcitabine given 22 hours after the radiolabeled peptide. However,administration of gemcitabine 19 hours prior to PT-RAIT resulted in asubstantial increase in survival (not shown).

Single PT-RAIT (0.25 mCi) combined with cetuximab (1 mg weekly ip; 7weeks) or with cetuximab+GEM (6 mg weekly×3) in animals bearing CaPan1showed the GEM+cetuximab combination with PT-RAIT providing a betterinitial response (FIG. 14), but the response associated with justcetuximab alone added to PT-RAIT was encouraging (FIG. 14), since it wasas good or better than PT-RAIT+GEM. Because the overall survival in thisstudy was excellent, with only 2 tumors in each group progressingto >2.0 cm3 after 24 weeks when the study was terminated, these resultsindicate a potential role for cetuximab when added to PT-RAIT.

Example 21 Effect of Fractionated Pretargeted Radioimmunotherapy(PT-RAIT) for Pancreatic Cancer Therapy

We evaluated fractionated therapy with ⁹⁰Y-DOTA-di-HSG peptide (IMP-288)and TF10. Studies using TF10 and radiolabeled IMP-288 were performed innude mice bearing s.c. CaPan1 human pancreatic cancer xenografts,0.32-0.54 cm³. For therapy, TF10-pretargeted ⁹⁰Y-IMP-288 was given [A]once (0.6mCi on wk 0) or [B] fractionated (0.3 mCi on wks 0 and 1), [C](0.2 mCi on wks 0, 1 and 2) or [D] (0.2 mCi on wks 0, 1 and 4).

Tumor regression (>90%) was observed in the majority of mice, 9/10,10/10, 9/10 and 8/10 in groups [A], [B], [C] and [D], respectively. Ingroup [A], maximum tumor regression in 50% of the mice was reached at3.7 wks, compared to 6.1, 8.1 and 7.1 wks in [B], [C] and [D],respectively. Some tumors showed regrowth. At week 14, the besttherapeutic response was observed in the fractionated group (2×0.3 mCi),with 6/10 mice having no tumors (NT) compared to 3/10 in the 3×0.2 mCigroups and 1/10 in the 1×0.6mCi group. No major body weight loss wasobserved. Fractionated PT-RAIT provides another alternative for treatingpancreatic cancer with minimum toxicity.

Example 22 ⁹⁰Y-hPAM4 Radioimmunotherapy (RAIT) Plus RadiosensitizingGemcitabine (GEM) Treatment in Advanced Pancreatic Cancer (PC)

⁹⁰Y-hPAM4, a humanized antibody highly specific for PC, showed transientactivity in patients with advanced disease, and GEM enhanced RAIT inpreclinical studies. This study evaluated repeated treatment cycles of⁹⁰Y-hPAM4 plus GEM in patients with untreated, unresectable PC. The⁹⁰Y-dose was escalated by cohort, with patients repeating 4-wk cycles(once weekly 200 mg/m² GEM, ⁹⁰Y-hPAM4 once-weekly wks 2-4) untilprogression or unacceptable toxicity. Response assessments used CT,FDG-PET, and CA19.9 serum levels.

Of 8 patients (3F/5M, 56-72 y.o.) at the 1^(st) 2 dose levels (6.5 and9.0 mCi/m² ⁹⁰Y-hPAM4×3), hematologic toxicity has been transient Grade1-2. Two patients responded to initial treatment with FDG SUV and CA19.9decreases, and lesion regression by CT. Both patients continue in goodperformance status now after 9 and 11 mo. and after a total of 3 and 4cycles, respectively, without additional toxicity. A 3^(rd) patient witha stable response by PET and CT and decreases in CA19.9 levels afterinitial treatment is now undergoing a 2nd cycle. Four other patients hadearly disease progression and the remaining patient is still beingevaluated. Dose escalation is continuing after fractionated RAIT with⁹⁰Y-hPAM4 plus low-dose gemcitabine demonstrated therapeutic activity atthe initial ⁹⁰Y-dose levels, with minimal hematologic toxicity, evenafter 4 therapy cycles.

Example 23 Early Detection of Pancreatic Carcinoma Using Mab-PAM4 and InVitro Immunoassay

Immunohistochemistry studies were performed with PAM4 antibody. Resultsobtained with stained tissue sections showed no reaction of PAM4 withnormal pancreatic ducts, ductules and acinar tissues (not shown). Incontrast, use of the MA5 antibody applied to the same tissue samplesshowed diffuse positive staining of normal pancreatic ducts and acinartissue (not shown). In tissue sections with well differentiated ormoderately differentiated pancreatic adenocarcinoma, PAM4 staining waspositive, with mostly cytoplasmic staining but intensification of at thecell surface. Normal pancreatic tissue in the same tissue sections wasunstained.

Table 11 shows the results of immunohistochemical analysis with PAM4 MAbin pancreatic adenocarcinoma samples of various stages ofdifferentiation. Overall, there was an 87% detection rate for allpancreatic cancer samples, with 100% detection of well differentiatedand almost 90% detection of moderately differentiated pancreaticcancers.

TABLE 11 PAM4 Labeling Pattern Cancer n Focal Diffuse Total Well Diff.13 2 11  13 (100%) Moderately Diff. 24 6 15 21 (88%) Poorly Diff. 18 5 914 (78%) Total 55 13 35 48 (87%)

Table 12 shows that PAM4 immunohistochemical staining also detected avery high percentage of precursor lesions of pancreatic cancer,including PanIn-1A to PanIN-3, IPMN (intraductal papillary mucinousneoplasms) and MCN (mucinous cystic neoplasms). Overall, PAM4 stainingdetected 89% of all pancreatic precursor lesions. These resultsdemonstrate that PAM4 antibody-based immunodetection is capable ofdetecting almost 90% of pancreatic cancers and precursor lesions by invitro analysis. PAM4 expression was observed in the earliest phases ofPanIN development. Intense staining was observed in IPMN and MCN samples(not shown). The PAM4 epitope was present at high concentrations(intense diffuse stain) in the great majority of pancreaticadenocarcinomas. PAM4 showed diffuse, intense reactivity with theearliest stages of pancreatic carcinoma precursor lesions, includingPanIN-1, IPMN and MCN, yet was non-reactive with normal pancreatictissue. Taken together, these results show that diagnosis and/ordetection with the PAM4 antibody is capable of detecting, with very highspecificity, the earliest stages of pancreatic cancer development.

TABLE 12 PAM4 Labeling Pattern n Focal Diffuse Total PanIn-1A 27 9 15 24(89%) PanIn-1B 20 4 16  20 (100%) PanIn-2 11 6 4 10 (91%) PanIn-3 5 2 0 2 (40%) Total PanIn 63 21 35 56 (89%) IPMN 36 6 25 31 (86%) MCN 27 3 2225 (92%)

An enzyme based immunoassay for PAM4 antigen in serum samples wasdeveloped. FIG. 15 shows the results of differential diagnosis usingPAM4 immunoassay for pancreatic cancer versus normal tissues and othertypes of cancer. The results showed a sensitivity of detection ofpancreatic cancer of 77.4%, with a specificity of detection of 94.3%,comparing pancreatic carcinoma (n=53) with all other specimens (n=233),including pancreatitis and breast, ovarian and colorectal cancer andlymphoma. The data of FIG. 15 are presented in tabular form in Table 13.

TABLE 13 PAM4-Reactive Mucin in Patient Sera # Positive n Mean SD MedianRange (%) Normal 43 0.1 0.3 0.0 0-2.0  0 (0) Pancreatitis 87 3.0 11.50.0 0-63.6 4 (5) Pancreatic CA 53 171 317 31.7  0-1000 41 (77)Colorectal CA 36 3.3 7.7 0.0 0-37.8  5 (14) Breast CA 30 3.7 10.1 0.00-53.5 2 (7) Ovarian CA 15 1.8 4.3 0.0 0-16.5 1 (7) Lymphoma 19 12.344.2 0.0 0-194  1 (5)

An ROC curve (not shown) was constructed with the data from Table 13.Examining a total of 283 patients, including 53 with pancreaticcarcinoma, and comparing the presence of circulating PAM4 antigen inpatients with pancreatic cancer to all other samples, the ROC curveprovided an AUC of 0.88±0.03 (95% ci, 0.84-0.92) with a P value<0.0001,a highly significant difference for discrimination of pancreaticcarcinoma from non-pancreatic carcinoma samples. Comparing pancreatic CAwith other tumors and normal tissue, the PAM4 based serum assay showed asensitivity of 77% and a specificity of 95%.

A comparison was made of PAM4 antigen concentration in serum samplesfrom normal patients, “early” (stage 1) pancreatic carcinoma and allpancreatic carcinoma samples. The specimens included 13 sera fromhealthy volunteers, 12 sera from stage-1, 13 sera from stage-2 and 25sera from stage-3/4 (advanced) pancreatic carcinoma. A cutoff value of8.8 units/ml (red line) was used, as determined by ROC curve statisticalanalysis. The frequency distribution of PAM4 antigen concentration isshown in FIG. 16, which shows that 92% of “early” stage-1 pancreaticcarcinomas were above the cutoff line for diagnosis of pancreaticcancer. An ROC curve for the PAM4 based assay is shown in FIG. 17, whichdemonstrates a sensitivity of 81.6% and specificity of 84.6% for thePAM4 assay in detection of pancreatic cancer.

These results confirm that an enzyme immunoassay based on PAM4 antibodybinding can detect and quantitate PAM4-reactive antigen in the serum ofpancreatic carcinoma patients. The immunoassay demonstrates highspecificity and sensitivity for pancreatic carcinoma. The majority ofpatients with stage 1 disease were detectable using the PAM4immunoassay.

In conclusion, an immunohistology procedure employing PAM4 antibodyidentified approximately 90% of invasive pancreatic carcinoma and itsprecursor lesions, PanIN, IPMN and MCN. A PAM4 based enzyme immunoassayto quantitate PAM4 antigen in human patient sera showed high sensitivityand specificity for detection of early pancreatic carcinoma. Due to thehigh specificity of PAM4 for pancreatic carcinoma, the mucin biomarkercan also serve as a target for in vivo targeting of imaging andtherapeutic agents. ImmunoPET imaging for detection of “early”pancreatic carcinoma is of use for the early diagnosis of pancreaticcancer, when it can be more effectively treated. Use ofradioimmunotherapy with a humanized PAM4 antibody construct, preferablyin combination with a radiosensitizing agent, is of use for thetreatment of pancreatic cancer.

Example 24 PEGylated DNL Constructs

In certain embodiments, it may be preferred to prepare constructscomprising PEGylated forms of antibody or immunoconjugate. SuchPEGylated constructs may be prepared by the DNL technique.

In a preferred method, the effector moiety to be PEGylated, such ashPAM4 Fab, is linked to a DDD sequence to generate the DDD module. A PEGreagent of a desirable molecular size is derivatized with acomplementary AD sequence and the resulting PEG-AD module is combinedwith the DDD module to produce the PEGylated conjugate that consists ofa single PEG tethered site-specifically to two copies of the Fab orother effector moiety via the disulfide bonds formed between DDD and AD.The PEG reagents may be capped at one end with a methoxy group (m-PEG),can be linear or branched, and may contain one of the followingfunctional groups: propionic aldehyde, butyric aldehyde,ortho-pyridylthioester (OPTE), N-hydroxysuccinimide (NHS),thiazolidine-2-thione, succinimidyl carbonate (SC), maleimide, orortho-pyridyldisulfide (OPPS). Among the effector moieties that may beof interest for PEGylation are enzymes, cytokines, chemokines, growthfactors, peptides, aptamers, hemoglobins, antibodies and antibodyfragments. The method is not limiting and a wide variety of agents maybe PEGylated using the disclosed methods and compositions. PEG ofvarious sizes and derivatized with a variety of reactive moieties may beobtained from commercial sources as discussed in more detail below.

Generation of PEG-AD2 Modules

IMP350: (SEQ ID NO: 41) CGQIEYLAKQIVDNAIQQAGC(SS-tbu)-NH₂ 

IMP350, incorporating the sequence of AD2, was made on a 0.1 mmol scalewith Sieber Amide resin using Fmoc methodology on a peptide synthesizer.Starting from the C-terminus the protected amino acids used wereFmoc-Cys(t-Buthio)-OH, Fmoc-Gly-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH,Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH, Fmoc-Ala-OH, Fmoc-Asn(Trt)-OH,Fmoc-Asp(OBut)-OH, Fmoc-Val-OH, Fmoc-Ile-OH, Fmoc-Gln(Trt)-OH,Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH, Fmoc-Leu-OH, Fmoc-Tyr(But)-OH,Fmoc-Glu(OBut)-OH, Fmoc-Ile-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gly-OH andFmoc-Cys(Trt)-OH. The peptide was cleaved from the resin and purified byreverse phase (RP)-HPLC.

Synthesis of PEG₂₀-IMP350

IMP350 (0.0104 g) was mixed with 0.1022 g of mPEG-OPTE (20 kDa, NEKTAR®Therapeutics) in 7 mL of 1 M Tris buffer at pH 7.81. Acetonitrile, 1 mL,was then added to dissolve some suspended material. The reaction wasstirred at room temperature for 3 h and then 0.0527 g of TCEP was addedalong with 0.0549 g of cysteine. The reaction mixture was stirred for1.5 h and then purified on a PD-10 desalting column, which wasequilibrated with 20% methanol in water. The sample was eluted, frozenand lyophilized to obtain 0.0924 g of crude PEG₂₀-IMP350 (MH+ 23508 byMALDI).

Synthesis of IMP362 (PEG₂₀-IMP360)

IMP360: (SEQ ID NO: 42) CGQIEYLAKQIVDNAIQQAGC(SS-tbu)G-EDANS MH⁺ 2660

IMP 360, incorporating the AD2 sequence, was synthesized on a 0.1 mmolscale with Fmoc-Gly-EDANS resin using Fmoc methodology on a peptidesynthesizer. The Fmoc-Gly-OH was added to the resin manually using 0.23g of Fmoc-Gly-OH, 0.29 g of HATU, 26 μL of DIEA, 7.5 mL of DMF and 0.57g of EDANS resin (NOVABIOCHEM®). The reagents were mixed and added tothe resin. The reaction was mixed at room temperature for 2.5 hr and theresin was washed with DMF and IPA to remove the excess reagents.Starting from the C-terminus the protected amino acids used wereFmoc-Cys(t-Buthio)-OH, Fmoc-Gly-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH,Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH, Fmoc-Ala-OH, Fmoc-Asn(Trt)-OH,Fmoc-Asp(OBut)-OH, Fmoc-Val-OH, Fmoc-Ile-OH, Fmoc-Gln(Trt)-OH,Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH, Fmoc-Leu-OH, Fmoc-Tyr(But)-OH,Fmoc-Glu(OBut)-OH, Fmoc-Ile-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gly-OH andFmoc-Cys(Trt)-OH. The peptide was cleaved from the resin and purified byRP-HPLC.

For synthesis of IMP362, IMP360 (0.0115 g) was mixed with 0.1272 g ofmPEG-OPTE (20 kDa, NEKTAR® Therapeutics) in 7 mL of 1 M tris buffer, pH7.81. Acetonitrile (1 mL) was then added to dissolve some suspendedmaterial. The reaction was stirred at room temperature for 4 h and then0.0410 g of TCEP was added along with 0.0431 g of cysteine. The reactionmixture was stirred for 1 h and purified on a PD-10 desalting column,which was equilibrated with 20% methanol in water. The sample waseluted, frozen and lyophilized to obtain 0.1471 g of crude IMP362 (MH+23713).

Synthesis of IMP413 (PEG₃₀-IMP360)

For synthesis of IMP 413, IMP 360 (0.0103 g) was mixed with 0.1601 g ofmPEG-OPTE (30 kDa, NEKTAR® Therapeutics) in 7 mL of 1 M tris buffer atpH 7.81. Acetonitrile (1 mL) was then added to dissolve some suspendedmaterial. The reaction was stirred at room temperature for 4.5 h andthen 0.0423 g of TCEP was added along with 0.0473 g of cysteine. Thereaction mixture was stirred for 2 h followed by dialysis for two days.The dialyzed material was frozen and lyophilized to obtain 0.1552 g ofcrude IMP413 (MH⁺34499).

Synthesis of IMP421

IMP 421 (SEQ ID NO: 43)Ac-C-PEG₃-C(S-tBu)GQIEYLAKQIVDNAIQQAGC(S-tBu)G-NH₂

The peptide IMP421, MH⁺2891 was made on NOVASYN® TGR resin (487.6 mg,0.112 mmol) by adding the following amino acids to the resin in theorder shown: Fmoc-Gly-OH, Fmoc-Cys(t-Buthio)-OH, Fmoc-Gly-OH,Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH,Fmoc-Ala-OH, Fmoc-Asn(Trt)-OH, Fmoc-Asp(OBut)-OH, Fmoc-Val-OH,Fmoc-Ile-OH, Fmoc-Gln(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH,Fmoc-Leu-OH, Fmoc-Tyr(But)-OH, Fmoc-Glu(OBut)-OH, Fmoc-Ile-OH,Fmoc-Gln(Trt)-OH, Fmoc-Gly-OH, Fmoc-Cys(t-Buthio)-OH, Fmoc-NH-PEG₃-COOH,Fmoc-Cys(Trt)-OH. The N-terminal amino acid was protected as an acetylderivative. The peptide was then cleaved from the resin and purified byRP-HPLC to yield 32.7 mg of a white solid.

Synthesis of IMP457

IMP 421 (SEQ ID NO:43, incorporating the sequence of AD2, wassynthesized by standard chemical means. To a solution of 15.2 mg (5.26μmol) IMP 421 (F.W. 2890.50) and 274.5 mg (6.86 μmol) mPEG2-MAL-40K in 1mL of acetonitrile was added 7 mL 1 M Tris pH 7.8 and allowed to reactat room temperature for 3 h. The excess mPEG2-MAL-40K was quenched with49.4 mg L-cysteine, followed by S—S-tBu deprotection over one hour with59.1 mg TCEP. The reaction mixture was dialyzed overnight at 2-8° C.using two 3-12 mL capacity 10K SLIDE-A-LYZER® dialysis cassettes (4 mlinto each cassette) into 5 L of 5 mM ammonium acetate, pH 5.0. Threemore 5 L buffer changes of 5 mM ammonium acetate, pH 5.0 were made thenext day with each dialysis lasting at least 2½ h. The purified product(19.4 mL) was transferred into two 20 mL scintillation vials, frozen andlyophilized to yield 246.7 mg of a white solid. MALDI-TOF gave resultsof mPEG2-MAL-40K 42,982 and IMP-457 45,500.

Example 25 Generation of PEGylated hPAM4 by DNL

A DNL structure is prepared having two copies of hPAM4 Fab coupled to a20 kDa PEG. A DNL reaction is performed by the addition of reduced andlyophilized IMP362 in 10-fold molar excess to hPAM4 Fab-DDD2 in 250 mMimidazole, 0.02% Tween 20, 150 mM NaCl, 1 mM EDTA, 50 mM NaH₂PO₄, pH7.5. After 6 h at room temperature in the dark, the reaction mixture isdialyzed against CM Loading Buffer (150 mM NaCl, 20 mM NaAc, pH 4.5) at4° C. in the dark. The solution is loaded onto a 1-mL Hi-Trap CM-FFcolumn (AMERSHAM®), which is pre-equilibrated with CM Loading buffer.After sample loading, the column is washed with CM loading buffer tobaseline, followed by washing with 15 mL of 0.25 M NaCl, 20 mM NaAc, pH4.5. The PEGylated hPAM4 is eluted with 12.5 mL of 0.5 M NaCl, 20 mMNaAc, pH 4.5.

The conjugation process is analyzed by SDS-PAGE with Coomassie bluestaining. Under non-reducing conditions, the Coomassie blue-stained gelreveals the presence of a major band in the reaction mixture, which isabsent in the unbound or 0.25 M NaCl wash fraction, but evident in the0.5 M NaCl fraction. Fluorescence imaging, which is used to detect theEDANS tag on IMP362, demonstrates that the band contains IMP362 and thepresence of excess IMP362 in the reaction mixture and the unboundfraction. The DNL reaction results in the site-specific and covalentconjugation of IMP362 with a dimer of hPAM4 Fab. Under reducingconditions, which breaks the disulfide linkage, the components of theDNL structures are resolved. The calculated MW of the (hPAM4 Fab)₂-PEGconstruct matches that determined by MALDI TOF. Overall, the DNLreaction results in a near quantitative yield of a homogeneous productthat is >90% pure after purification by cation-exchange chromatography.

Another DNL reaction is performed by the addition of reduced andlyophilized IMP457 in 10-fold molar excess to hPAM4 Fab-DDD2 in 250 mMimidazole, 0.02% Tween 20, 150 mM NaCl, 1 mM EDTA, 50 mM NaH₂PO₄, pH7.5. After 60 h at room temperature, 1 mM oxidized glutathione is addedto the reaction mixture, which is then held for an additional 2 h. Themixture is diluted 1:20 with CM Loading Buffer (150 mM NaCl, 20 mM NaAc,pH 4.5) and titrated to pH 4.5 with acetic acid. The solution is loadedonto a 1-mL Hi-Trap CM-FF column (AMERSHAM®), which is pre-equilibratedwith CM Loading Buffer. After sample loading, the column is washed withCM Loading Buffer to baseline, followed by washing with 15 mL of 0.25 MNaCl, 20 mM NaAc, pH 4.5. The PEGylated product is eluted with 20 mL of0.5 M NaCl, 20 mM NaAc, pH 4.5. The DNL construct is concentrated to 2mL and diafiltered into 0.4 M PBS, pH 7.4. The final PEGylated hPAM4Fab₂ construct is approximately 90% purity as determined by SDS-PAGE.

A DNL construct having two copies of hPAM4 Fab coupled to a 30 kDa PEGis prepared as described immediately above using IMP413 instead ofIMP362. The PEGylated hPAM4 Fab₂ DNL construct is purified as describedabove and obtained in approximately 90% purity. The PEGylated DNLconstructs may be used for therapeutic methods as described above fornon-PEGylated foams of hPAM4.

Example 26 Generation of DDD module based on Interferon (IFN)-α2b

The cDNA sequence for IFN-α2b was amplified by PCR, resulting in asequence comprising the following features, in which XbaI and BamHI arerestriction sites, the signal peptide is native to IFN-α2b, and 6 His isa hexahistidine tag (SEQ ID NO: 59): XbaI-Signal peptide-IFNα2b-6His-BamHI (6 His disclosed as SEQ ID NO: 59). The resulting secretedprotein consists of IFN-α2b fused at its C-terminus to a polypeptideconsisting of SEQ ID NO:44.

(SEQ ID NO: 44)KSHHHHHHGSGGGGSGGGCGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLR EARA

PCR amplification was accomplished using a full length human IFNα2b cDNAclone (INVITROGEN® Ultimate ORF human clone cat# HORF01Clone IDIOH35221) as a template and the following oligonucleotides as primers:

IFNA2 Xba I Left (SEQ ID NO: 45)5'-TCTAGACACAGGACCTCATCATGGCCTTGACCTTTGCTTTACTG G-3' IFNA2 BamHI right(SEQ ID NO: 46) 5'GGATCCATGATGGTGATGATGGTGTGACTTTTCCTTACTTCTTAAACTTTCTTGC-3'

The PCR amplimer was cloned into the PGEMT® vector (PROMEGA®). ADDD2-pdHL2 mammalian expression vector was prepared for ligation withIFN-α2b by digestion with XbaI and Bam HI restriction endonucleases. TheIFN-α2b amplimer was excised from PGEMT® with XbaI and Bam HI andligated into the DDD2-pdHL2 vector to generate the expression vectorIFN-α2b-DDD2-pdHL2.

IFN-α2b-DDD2-pdHL2 was linearized by digestion with SalI enzyme andstably transfected into Sp/EEE myeloma cells by electroporation (see,e.g., U.S. Pat. No. 7,537,930). Two clones were found to have detectablelevels of IFN-α2b by ELISA. One of the two clones, designated 95, wasadapted to growth in serum-free media without substantial decrease inproductivity. The clone was subsequently amplified with increasingmethotrexate (MTX) concentrations from 0.1 to 0.8 μM over five weeks. Atthis stage, it was sub-cloned by limiting dilution and the highestproducing sub-clone (95-5) was expanded. The productivity of 95-5 grownin shake-flasks was estimated to be 2.5 mg/L using commercial rIFN-α2b(CHEMICON®, IF007, Lot 06008039084) as a standard.

Clone 95-5 was expanded to 34 roller bottles containing a total of 20 Lof serum-free Hybridoma SFM with 0.8 μM MTX and allowed to reachterminal culture. The supernatant fluid was clarified by centrifugationand filtered (0.2 μM). The filtrate was diafiltered into 1× Bindingbuffer (10 mM imidazole, 0.5 M NaCl, 50 mM NaH₂PO₄, pH 7.5) andconcentrated to 310 mL in preparation for purification by immobilizedmetal affinity chromatography (IMAC). The concentrate was loaded onto a30-mL Ni-NTA column, which was washed with 500 mL of 0.02% Tween 20 in1× binding buffer and then 290 mL of 30 mM imidazole, 0.02% Tween 20,0.5 M NaCl, 50 mM NaH₂PO₄, pH 7.5. The product was eluted with 110 mL of250 mM imidazole, 0.02% Tween 20, 150 mM NaCl, 501nM NaH₂PO₄, pH 7.5.Approximately 6 mg of IFNα2b-DDD2 was purified.

The purity of IFN-α2b-DDD2 was assessed by SDS-PAGE under reducingconditions (not shown). IFN-α2b-DDD2 was the most heavily stained bandand accounted for approximately 50% of the total protein (not shown).The product resolved as a doublet with an M_(r) of ˜26 kDa, which isconsistent with the calculated MW of IFN-α2b-DDD2-SP (26 kDa). There wasone major contaminant with a M_(r) of 34 kDa and many faintcontaminating bands (not shown).

Example 27 Generation of hPAM4 Fab-(IFN-α2b)₂ by DNL

Creation of C—H-AD2-IgG-pdHL2 expression vectors.

The pdHL2 mammalian expression vector has been used to mediate theexpression of many recombinant IgGs. A plasmid shuttle vector wasproduced to facilitate the conversion of any IgG-pdHL2 vector into aC—H-AD2-IgG-pdHL2 vector. The gene for the Fc (CH2 and CH3 domains) wasamplified using the pdHL2 vector as a template and the oligonucleotidesFc BglII Left and Fc Bam-EcoRI Right as primers.

(SEQ ID NO: 47) 5'-AGATCTGGCGCACCTGAACTCCTG-3' Fc Bam-EcoRI Right(SEQ ID NO: 48) 5'-GAATTCGGATCCTTTACCCGGAGACAGGGAGAG-3'

The amplimer was cloned in the PGEMT® PCR cloning vector. The Fc insertfragment was excised from PGEMT® and ligated with AD2-pdHL2 vector togenerate the shuttle vector Fc-AD2-pdHL2.

Generation of hPAM4 IgG-AD2

To convert any IgG-pdHL2 expression vector to a C—H-AD2-IgG-pdHL2expression vector, an 861 bp BsrGI/NdeI restriction fragment is excisedfrom the former and replaced with a 952 bp BsrGI/NdeI restrictionfragment excised from the Fc-AD2-pdHL2 vector. BsrGI cuts in the CH3domain and NdeI cuts downstream (3′) of the expression cassette. Thismethod is used to generate a hPAM4 IgG-AD2 protein.

Generation of hPAM4 IgG-(IFN-α2b)₂ Construct

A DNL reaction is performed by the addition of reduced and lyophilizedhPAM4 IgG-AD2 to IFN-α2b-DDD2 in 250 mM imidazole, 0.02% Tween 20, 150mM NaCl, 1 mM EDTA, 50 mM NaH₂PO₄, pH 7.5. After 6 h at room temperaturein the dark, the reaction mixture is dialyzed against CM Loading Buffer(150 mM NaCl, 20 mM NaAc, pH 4.5) at 4° C. in the dark. The solution isloaded onto a 1-mL Hi-Trap CM-FF column (AMERSHAM®), which ispre-equilibrated with CM Loading buffer. After sample loading, thecolumn is washed with CM loading buffer to baseline, followed by washingwith 15 mL of 0.25 M NaCl, 20 mM NaAc, pH 4.5. The product is elutedwith 12.5 mL of 0.5 M NaCl, 20 mM NaAc, pH 4.5. The DNL reaction resultsin the site-specific and covalent conjugation of hPAM4 IgG with a dimerof IFN-α2b. Both the IgG and IFN-α2b moieties retain their respectivephysiological activities in the DNL construct. This technique may beused to attach any cytokine or other physiologically active protein orpeptide to hPAM4 for targeted delivery to pancreatic cancer or othercancers that express the PAM4 antigen.

Example 28 AD and DDD Sequence Variants

In certain preferred embodiments, the AD and DDD sequences incorporatedinto the DNL complexes comprise the amino acid sequences of AD2 (SEQ IDNO:36) and DDD2 (SEQ ID NO:34), as described above. However, inalternative embodiments sequence variants of the AD and/or DDD moietiesmay be utilized in construction of the cytokine-MAb DNL complexes. Thestructure-function relationships of the AD and DDD domains have been thesubject of investigation. (See, e.g., Burns-Hamuro et al., 2005, ProteinSci 14:2982-92; Carr et al., 2001, J Biol Chem 276:17332-38; Alto etal., 2003, Proc Natl Acad Sci USA 100:4445-50; Hundsrucker et al., 2006,Biochem J 396:297-306; Stokka et al., 2006, Biochem J 400:493-99; Goldet al., 2006, Mol Cell 24:383-95; Kinderman et al., 2006, Mol Cell24:397-408.)

For example, Kinderman et al. (2006) examined the crystal structure ofthe AD-DDD binding interaction and concluded that the human DDD sequencecontained a number of conserved amino acid residues that were importantin either dimer formation or AKAP binding, underlined in SEQ ID NO:33below. (See FIG. 1 of Kinderman et al., 2006.) The skilled artisan willrealize that in designing sequence variants of the DDD sequence, onewould desirably avoid changing any of the underlined residues, whileconservative amino acid substitutions might be made for residues thatare less critical for dimerization and AKAP binding.

Human DDD Sequence from Protein Kinase A

SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO:33)

Alto et al. (2003) performed a bioinformatic analysis of the AD sequenceof various AKAP proteins to design an RII selective AD sequence calledAKAP-IS (SEQ ID NO:35), with a binding constant for DDD of 0.4 nM. TheAKAP-IS sequence was designed as a peptide antagonist of AKAP binding toPKA. Residues in the AKAP-IS sequence where substitutions tended todecrease binding to DDD are underlined in SEQ ID NO:35 below.

AKAP-IS sequence (SEQ ID NO: 35) QIEYLAKQIVDNAIQQA

Similarly, Gold (2006) utilized crystallography and peptide screening todevelop a SuperAKAP-IS sequence (SEQ ID NO:49), exhibiting a five orderof magnitude higher selectivity for the RII isoform of PKA compared withthe RI isoform. Underlined residues indicate the positions of amino acidsubstitutions, relative to the AKAP-IS sequence, that increased bindingto the DDD moiety of RIIα. In this sequence, the N-terminal Q residue isnumbered as residue number 4 and the C-terminal A residue is residuenumber 20. Residues where substitutions could be made to affect theaffinity for RIIα were residues 8, 11, 15, 16, 18, 19 and 20 (Gold etal., 2006). It is contemplated that in certain alternative embodiments,the SuperAKAP-IS sequence may be substituted for the AKAP-IS AD moietysequence to prepare cytokine-MAb DNL constructs. Other alternativesequences that might be substituted for the AKAP-IS AD sequence areshown in SEQ ID NO:50-52. Substitutions relative to the AKAP-IS sequenceare underlined. It is anticipated that, as with the AKAP-IS sequenceshown in SEQ ID NO:49, the AD moiety may also include the additionalN-terminal residues cysteine and glycine and C-terminal residues glycineand cysteine.

SuperAKAP-IS (SEQ ID NO: 49) QIEYVAKQIVDYAIHQAAlternative AKAP sequences (SEQ ID NO: 50) QIEYKAKQIVDHAIHQA(SEQ ID NO: 51) QIEYHAKQIVDHAIHQA (SEQ ID NO: 52) QIEYVAKQIVDHAIHQA

Stokka et al. (2006) also developed peptide competitors of AKAP bindingto PKA, shown in SEQ ID NO:53-55. The peptide antagonists weredesignated as Ht31 (SEQ ID NO:53), RIAD (SEQ ID NO:54) and PV-38 (SEQ IDNO:55). The Ht-31 peptide exhibited a greater affinity for the RIIisoform of PKA, while the RIAD and PV-38 showed higher affinity for RI.

Ht31 (SEQ ID NO: 53) DLIEEAASRIVDAVIEQVKAAGAY RIAD (SEQ ID NO: 54)LEQYANQLADQIIKEATE PV-38 (SEQ ID NO: 55) FEELAWKIAKMIWSDVFQQC

Hundsrucker et al. (2006) developed still other peptide competitors forAKAP binding to PKA, with a binding constant as low as 0.4 nM to the DDDof the RII form of PKA. The sequences of various AKAP antagonisticpeptides is provided in Table 1 of Hundsrucker et al. (incorporatedherein by reference). Residues that were highly conserved among the ADdomains of different AKAP proteins are indicated below by underliningwith reference to the AKAP IS sequence (SEQ ID NO:35). The residues arethe same as observed by Alto et al. (2003), with the addition of theC-terminal alanine residue. (See FIG. 4 of Hundsrucker et al. (2006),incorporated herein by reference.) The sequences of peptide antagonistswith particularly high affinities for the RII DDD sequence are shown inSEQ ID NO:56-58.

AKAP-IS (SEQ ID NO: 35) QIEYLAKQIVDNAIQQA AKAP7δ-wt-pep (SEQ ID NO: 56)PEDAELVRLSKRLVENAVLKAVQQY AKAP7δ-L304T-pep (SEQ ID NO: 57)PEDAELVRTSKRLVENAVLKAVQQY AKAP7δ-L308D-pep (SEQ ID NO: 58)PEDAELVRLSKRDVENAVLKAVQQY

Carr et al. (2001) examined the degree of sequence homology betweendifferent AKAP-binding DDD sequences from human and non-human proteinsand identified residues in the DDD sequences that appeared to be themost highly conserved among different DDD moieties. These are indicatedbelow by underlining with reference to the human PKA RIIα DDD sequenceof SEQ ID NO:33. Residues that were particularly conserved are furtherindicated by italics. The residues overlap with, but are not identicalto those suggested by Kinderman et al. (2006) to be important forbinding to AKAP proteins.

(SEQ ID NO: 33) SHIQ IP P GL TELLQGYT V EVLR Q QP PDLVEFA VE YF TR L REAR A

The skilled artisan will realize that in general, those amino acidresidues that are highly conserved in the DDD and AD sequences fromdifferent proteins are ones that it may be preferred to remain constantin making amino acid substitutions, while residues that are less highlyconserved may be more easily varied to produce sequence variants of theAD and/or DDD sequences described herein.

The skilled artisan will realize that these and other amino acidsubstitutions in the antibody moiety or linker portions of the DNLconstructs may be utilized to enhance the therapeutic and/orpharmacokinetic properties of the resulting DNL constructs.

Example 29 In Vitro Detection of PAM4 Antigen in Human Serum

In certain embodiments, it is preferred to detect the presence of PAM4antigen and/or to diagnose the presence of pancreatic cancer in asubject by in vitro analysis of samples that can be obtained bynon-invasive techniques, such as blood, plasma or serum samples. Such exvivo analysis may be preferred, for example, in screening procedureswhere there is no a priori reason to believe that an individual has apancreatic tumor in a specific location.

Studies were initially performed using patient serum samples that hadbeen stored frozen for a number of years prior to analysis (Gold et al.,J. Clin Oncol 2006, 24:252-58). An in vitro enzyme immunoassay wasestablished with monoclonal antibody PAM4 as the capture reagent, and apolyclonal anti-mucin antibody as the probe. Patient sera were obtainedfrom healthy, adult patients with acute and chronic pancreatitis, andthose with pancreatic and other forms of cancer, and were measured forPAM4 antigen.

Methods

Reagents—

A human pancreatic mucin preparation was isolated from CaPan1, a humanpancreatic cancer grown as xenografts in athymic nude mice. Briefly, 1 gof tissue was homogenized in 10 mL of 0.1 M ammonium bicarbonatecontaining 0.5 M sodium chloride. The sample was then centrifuged toobtain a supernatant that was fractionated on a SEPHAROSE®-4B-CL columnwith the void volume material chromatographed on hydroxyapatite. Theunadsorbed fraction was dialyzed extensively against deionized water andthen lyophilized. A 1 mg/mL solution was prepared in 0.01 M sodiumphosphate buffer (pH, 7.2) containing 0.15 M sodium chloride(phosphate-buffered saline [PBS]), and used as the stock solution forthe immunoassay standards. A polyclonal, anti-mucin antiserum wasprepared by immunization of rabbits, as described previously (Gold etal., Cancer Res 43:235-38, 1983). An IgG fraction was purified andassessed for purity by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) and molecular-sieve high-performance liquidchromatography. Kits for quantitation of CA19-9 were purchased fromPANOMICS® Inc (Redwood City, Calif.).

Enzyme Immunoassay—

Sera were obtained from patients enrolled in institutional reviewboard-approved clinical trials conducted by the Garden State CancerCenter (Belleville, N.J.), as well as from the Eastern Division of theCooperative Human Tissue Network (National Cancer Institute [NCI],National Institutes of Health, Bethesda, Md.). To perform theimmunoassay, a 96-well polyvinyl plate was coated with 100 μL of PAM4antibody at 20 μg/mL in PBS with incubation at 4° C. overnight. On thenext morning, the capture antibody was removed from the plate. The wellswere then blocked by addition of 200 μL of a 1% (weight/volume [w/v])solution of casein-sodium salt in PBS and incubated overnight at 4° C.The casein was removed from the wells and the plate washed 5x with 250μL of PBS containing 0.05% (volume/volume [v/v]) Tween-20. The standard,or unknown specimen, was diluted 1:2 in 1% (w/v) casein in PBS, 100 ptwas added to the appropriate wells, and the plate incubated at 37° C.for 1.5 hours. At this time, the plate was washed five times withPBS-Tween-20 as mentioned already. The polyclonal rabbit anti-mucinantibody, diluted to 5 μg/mL in 0.1% (w/v) casein in PBS, was added toeach well, and the plate incubated for 1 hour at 37° C. The polyclonalantibody was then washed from the wells as described herein, andperoxidase-labeled donkey antirabbit IgG, at a 1:1000 dilution in 0.1%(w/v) casein in PBS, was added to the wells and incubated at 37° C. for1 hour. After washing the plate as already described, 100 μK of asolution consisting of ortho-phenylenediamine (0.8 mg/mL) and hydrogenperoxide (0.3% v/v in 0.1 M Tris-HCl [pH 8.0]), were added to the wells,and the plate was incubated at room temperature for 30 minutes in thedark. The reaction was stopped by the addition of 25 μL of 4.0 Nsulfuric acid, and the optical density read at a wavelength of 490 nmusing a SPECTRA-MAX® 250 spectrophotometer. CA19-9 determinations wereperformed according to the manufacturer's procedure. Standard curveswere generated with regression analyses performed to determineconcentrations of the unknown samples. Receiver operating characteristic(ROC) curves were generated by use of MED-CALC® statistical softwarepackage (version 7.5; MED-CALC®, Mariakerke Belgium).

Results

Development and Characterization of the PAM4-Based Immunoassay—

We chose to report our results in arbitrary units/mL based on an initialreference standard of mucin purified from xenografted CaPan1 humanpancreatic tumor. The lower limit of detection for the immunoassay was1.0 unit/mL, with saturation occurring at mucin concentrations above 100units/mL. A linear range was determined to be 1.5 units/mL to 25units/mL of antigen (not shown). Interassay (n=5) coefficients ofvariation (CV) were calculated for reference standards of 20 units/mL(CV=8.0%) and 8 units/mL (CV=3.8%). Mean recoveries were 17.5±2.8 and7.1±1.9 for the 20 and 8 units/mL standards, respectively.

Levels of PAM4-Reactive Mucin in Patient Specimens—

Sera from a total of 283 patients, including 53 with pancreatic cancer,were examined for the presence of PAM4 antigen. The frequencydistribution of serum mucin concentrations for the varying diseasegroups was determined (FIG. 16). The receiver operator characteristic(ROC) curve was calculated (not shown), and the area under the curve(AUC) determined to be 0.88±0.03 (95% CI, 0.84 to 0.92) with P<0.0001(see Example 23), a highly significant difference for discrimination ofpancreatic cancer from nonpancreatic cancer specimens. At a cutoff valueof 10.2 units/mL, the sensitivity and specificity were calculated to be77% and 95% (Example 23), respectively, with a positive diagnosticlikelihood ratio (+DLR) of 13.7, as compared with healthy, benign, andnonpancreatic cancer groups.

The data presented in Table 13 showed that the median and mean valuesfor the pancreatic cancer group were more than 10-fold greater than forall of the other groups, even though the overwhelming majority of thenonpancreatic cancer cases were late-stage disease. Of 53 pancreaticcancer patients, 41 (77%) were positive at a cutoff value of 10.2units/mL. At this same cutoff value, none of the healthy specimens werepositive, and only four (5%) of 87 pancreatitis patients were positive.ROC curve analyses demonstrated high specificity with significantdifferences for the discrimination of pancreatic cancer from normal andpancreatitis patient groups (not shown).

Comparison of PAM4 and CA19.9 Immunonassays—

Of the 53 pancreatic cancer specimens, only 41 were assessable for bothPAM4-reactive mucin and CA 19-9 because of insufficient volume ofcertain samples. Of these, 24 (59%) were considered positive for CA19-9(at a cutoff of 35 units/mL). As with the PAM4 immunoassay, none of thehealthy specimens were positive for CA19-9. However, of the 87pancreatitis samples, CA19-9 was positive in 37% (not shown). ROCanalyses for discrimination of pancreatic cancer from pancreatitis serumspecimens provided an AUC of 0.67±0.05 (95% CI, 0.58 to 0.75), with aspecificity of 63% and a +DLR of 1.6 for the CA19-9 test (not shown).Statistical analyses for PAM4-reactive mucin in this same subset ofpancreatic cancer and pancreatitis sera differed little from the groupanalyses discussed earlier; sensitivity for this subset was slightlyreduced (71%), but specificity remained high (96%), as did the +DLR(15.4) (not shown). There was no correlation between PAM4 and CA19-9values. Two of the four PAM4-positive pancreatitis specimens were alsopositive for CA19-9. A direct pair-wise comparison of the ROC curvesresulted in a statistically significant difference (P<0.003), with thePAM4 immunoassay demonstrating a superior sensitivity and specificityfor discrimination of patients with pancreatic cancer from those withpancreatitis (not shown).

The results obtained with serum samples frozen at −80° C. and frozen fora period of years before analysis were not initially reproducible withfresher serum samples. Immunoassay with PAM4 antibody performed on freshserum samples obtained from individuals with known pancreatic cancer, orfresh serum samples spiked with pancreatic cancer mucin, routinelyresulted in false negative results. It was observed that the frozen andstored samples, after thawing, usually separated into lipid and aqueouscomponents. The lipid component was removed by centrifugation before thePAM4 immunoassay was performed on the remaining aqueous component. Inthe initial studies with fresher serum samples, the serum did notseparate and the immunoassay was run on whole serum.

To reproduce the effects of freezing and long-term storage, the fresherserum samples were subject to an organic phase extraction to removeserum lipids and other hydrophobic components. Although the phaseextraction was performed with butanol, the skilled artisan will realizethat the technique is not so limited and may be performed withalternative organic solvents known in the art. Exemplary organicsolvents known in the art include other alcohols that are not misciblewith water, chloroform, hexane, benzene, DMF (dimethyl formamide), DMSO(dimethyl sulfoxide) and ether.

To perform the organic phase extraction of serum samples, 300 μL ofserum was placed in a 1.5 mL microcentrifuge tube, then 300 μL ofn-Butanol was added, the tube closed tightly and vigorously vortexedseveral times during a 5 min extraction period. At the end of theextraction, the tubes were opened and 300 μL of chloroform was added.The tubes were closed tightly, the tube was vigorously vorteed and thenspun in a tabletop centrifuge at high speed for 5 min. The tubes wereopened and 200 μL of the upper aqueous phase was removed to a cleantube. An equal volume of immunoassay diluent (2% casein) was added (1:2dilution of the unknown serum) and used as antigen in the immunoassayprotocol described above. Using the organic phase extraction, resultsfor PAM4 antigen detection and pancreatic cancer diagnosis were obtainedthat were equivalent to those seen with samples subjected to long termfreezing and storage described above (not shown).

The interference of an organic component with PAM4-based immunoassayseen with fresh serum samples was not observed with PAM4 immunohistologyon formalin-fixed, paraffin-embedded tissue sections, which aretypically processed with an organic solvent extraction prior toimmunoassay. The interfering component appears to be limited indistribution to serum and is not observed to interfere with PAM4antibody binding to solid pancreatic cancer tumors in situ.

Example 30 Preparation and Assay of Cross-Blocking Antibodies to PAM4

Tumors of xenografted RIP1 human pancreatic carcinoma are grown in nudemice and harvested. The human pancreatic cancer mucins are extractedaccording to Gold et al. (Int J Cancer 1994, 15:204-10) and used toimmunize mice according to standard protocols (Harlow and Lane,Antibodies: A Laboratory Manual, Ch. 5, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.) Antibody producing hybridoma cells areprepared from the immunized mice and screened for binding to humanpancreatic cancer mucin extracts. Positive clones are expanded and themonoclonal antibodies are tested for cross-blocking activity againstcPAM4 using competitive binding assays as described in Example 1.Cross-blocking antibodies against cPAM4 are identified by competitionfor binding to human pancreatic cancer mucin.

It will be apparent to those skilled in the art that variousmodifications and variations can be made to the products, compositions,methods and processes of this invention. Thus, it is intended that thepresent invention cover such modifications and variations, provided theycome within the scope of the appended claims and their equivalents.

1. A method of treating pancreatic cancer, comprising administering to asubject with pancreatic cancer an antibody construct comprising at leastone humanized or human anti-pancreatic cancer antibody orantigen-binding fragment thereof that can bind to a linear peptidecomprising the amino acid sequence WTWNITKAYPLP (SEQ ID NO:29) or to acyclic peptide comprising the amino acid sequence ACPEWWGTTC (SEQ IDNO:30).
 2. The method of claim 1, wherein the anti-pancreatic cancerantibody or fragment thereof binds to pancreatic cancer cells and doesnot bind to normal pancreatic tissue or pancreatitis or benignpancreatic tissue.
 3. The method of claim 2, wherein the anti-pancreaticcancer antibody binds to PanIN-1A, PanIN-1B, PanIN-2, invasivepancreatic adenocarcinoma, pancreatic carcinoma, mucinous cyst neoplasms(MCN), intrapancreatic mucinous neoplasms (IPMN) and intraductalpapillary mucinous neoplasia.
 4. The method of claim 3, wherein theanti-pancreatic cancer antibody or antigen-binding fragment thereofbinds to 80% or more of human invasive pancreatic adenocarcinoma,intraductal papillary mucinous neoplasia, PanIN-1A, PanIN-1B andPanIN-2.
 5. The method of claim 1, wherein the antibody construct is aDNL (dock and lock) construct comprising: a) at least one humanized orhuman anti-pancreatic cancer antibody or antigen-binding fragmentthereof that can bind to a linear peptide comprising the amino acidsequence WTWNITKAYPLP (SEQ ID NO:29) or to a cyclic peptide comprisingthe amino acid sequence ACPEWWGTTC (SEQ ID NO:30); and b) a secondantibody or antigen-binding fragment thereof attached to the at leastone chimeric, humanized or human anti-pancreatic cancer antibody orantigen-binding fragment thereof.
 6. The method of claim 5, wherein eachhumanized or human anti-pancreatic cancer antibody fragment is a Fabfragment attached to a DDD2 (SEQ ID NO:34) sequence and the secondantibody fragment is a Fab fragment attached to an AD2 (SEQ ID NO:36)sequence and wherein a dimer of the DDD2 sequence binds to the AD2sequence to form the DNL construct.
 7. The method of claim 5, whereinthe second antibody or antigen-binding fragment thereof binds to a tumorassociated antigen (TAA) or a hapten.
 8. The method of claim 7, whereinthe TAA is selected from the group consisting of a Lewis antigen, Le(y),carcinoembryonic antigen (CEACAM5), CEACAM6, colon-specific antigen-p(CSAp), MUC-1, MUC-2, MUC-3, MUC-4, MUC-5ac, MUC-16, MUC-17, HLA-DR,CD40, CD74, CD80, CD138, HER2/neu, EGFR, EGP-1, EGP-2, an angiogenesisfactor, VEGF, P1GF, insulin-like growth factor, carbonic anhydrase IX,tenascin, platelet-derived growth factor, IL-6, an oncogene product,bcl-2, Kras, p53, cMET and a tumor necrosis factor.
 9. The method ofclaim 5, wherein the second antibody is selected from the groupconsisting of CA19.9, DUPAN2, SPAN1, Nd2, B72.3, CC49, hA20, hA19,hIMMU31, hLL1, hLL2, hMu-9, hL243, hMN-3, hMN-14, hMN-15, hRS7 and hR1.10. The method of claim 7, wherein the hapten is HSG(histamine-succinyl-glycine) or In-DTPA (diethylenetriaminepentaaceticacid).
 11. The method of claim 10, further comprises administering tothe subject a targetable construct comprising at least one HSG orIn-DTPA moiety, wherein the targetable construct is attached to at leastone therapeutic agent.
 12. The method of claim 1, wherein theanti-pancreatic cancer antibody or antigen-binding fragment thereof is anaked antibody or fragment thereof.
 13. The method of claim 12, furthercomprising administering at least one therapeutic agent to theindividual.
 14. The method of claim 1, wherein the anti-pancreaticcancer antibody or antigen-binding fragment thereof is conjugated to atleast one therapeutic agent.
 15. The method of claim 14, wherein thetherapeutic agent is selected from the group consisting of aradionuclide, an immunomodulator, a hormone, a hormone antagonist, anenzyme, an anti-sense oligonucleotide, an siRNA, an enzyme inhibitor, aphotoactive therapeutic agent, a cytotoxic agent, a drug, a pro-drug, atoxin, an angiogenesis inhibitor and a pro-apoptotic agent.
 16. Themethod of claim 15, wherein the drug is selected from the groupconsisting of nitrogen mustards, ethylenimine derivatives, alkylsulfonates, nitrosoureas, gemcitabine, triazenes, folic acid analogs,anthracyclines, taxanes, COX-2 inhibitors, pyrimidine analogs, purineanalogs, antibiotics, enzyme inhibitors, epipodophyllotoxins, platinumcoordination complexes, vinca alkaloids, substituted ureas, methylhydrazine derivatives, adrenocortical suppressants, hormone antagonists,endostatin, taxols, camptothecins, SN-38, doxorubicin, antimetabolites,alkylating agents, antimitotics, anti-angiogenic agents, tyrosine kinaseinhibitors, mTOR inhibitors, heat shock protein (HSP90) inhibitors,proteosome inhibitors, HDAC inhibitors, pro-apoptotic agents,methotrexate and CPT-11.
 17. The method of claim 15, wherein the toxinis selected from the group consisting of ricin, abrin, alpha toxin,saporin, ribonuclease (RNase), DNase I, Staphylococcal enterotoxin-A,pokeweed antiviral protein, gelonin, diphtheria toxin, Pseudomonasexotoxin, and Pseudomonas endotoxin.
 18. The method of claim 15, whereinthe immunomodulator is selected from the group consisting of a cytokine,a stem cell growth factor, a lymphotoxin, a hematopoietic factor, acolony stimulating factor (CSF), an interleukin (IL), an interferon(IFN), a stem cell growth factor, erythropoietin, thrombopoietin, tumornecrosis factor (TNF), granulocyte-colony stimulating factor (G-CSF),granulocyte macrophage-colony stimulating factor (GM-CSF), interferon-α,interferon-β, interferon-γ and the stem cell growth factor designated“S1 factor”.
 19. The method of claim 18, wherein the cytokine isselected from the group consisting of human growth hormone, N-methionylhuman growth hormone, bovine growth hormone, parathyroid hormone,thyroxine, insulin, proinsulin, relaxin, prorelaxin, glycoproteinfollicle stimulating hormone (FSH), thyroid stimulating hormone (TSH),luteinizing hormone (LH), placenta growth factor (P1GF), hepatic growthfactor, prostaglandin, fibroblast growth factor, prolactin, placentallactogen, OB protein, tumor necrosis factor-α, tumor necrosis factor-β,mullerian-inhibiting substance, mouse gonadotropin-associated peptide,inhibin, activin, vascular endothelial growth factor, integrin,thrombopoietin (TPO), NGF-β, platelet-growth factor, TGF-α, TGF-β,insulin-like growth factor-I, insulin-like growth factor-II,erythropoietin (EPO), osteoinductive factors, interferon-α,interferon-β, interferon-γ, macrophage-CSF (M-CSF), IL-1, IL-1α, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13,IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, IL-25, LIF, FLT-3,angiostatin, thrombospondin, endostatin, TNF-α and LT (lymphotoxin). 20.The method of claim 15, wherein the radionuclide is selected from thegroup consisting of ¹¹¹In, 177Lu, ²¹²Bi, ²¹³Bi, 211At, ⁶²Cu, ⁶⁷Cu, ⁹⁰Y,¹²⁵I, ¹³¹I, ³²P, ³³P, ⁴⁷Se, ¹¹¹Ag, ⁶⁷Ga, ¹⁴³Pr, ¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁶Dy,¹⁶⁶Ho, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re, ²¹²Pb, ²²³Ra, ²²⁵Ac, ⁵⁹Fe, ⁷⁵Tc, ⁷⁷As, ⁸⁹Sr,⁹⁹Mo, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹⁴³Pr, ¹⁴⁹Pm, ¹⁶⁹Er, ¹⁹⁴Ir, ¹⁹⁸Au, ¹⁹⁹Au, ²¹¹Pb,⁵⁸Co, ^(80m)Br, ^(99m)Tc, ^(103m)Rh, ¹⁰⁹Pt, ¹¹⁹Sb, ^(189m)Os, ¹⁹²Ir,²¹⁹Rn, ²¹⁵Po, ²²¹Fr, ²¹⁷At, ²⁵⁵Fm, ¹¹C, ¹³N, ¹⁵O, ⁷⁵Br, ²²⁴Ac, ¹²⁶I,¹³³I, ⁷⁷Br, ⁹⁵Ru, ⁹⁷Ru, ¹⁰³Ru, ¹⁰⁵Ru, ¹⁰⁷Hg, ²⁰³Hg, ^(121m)Te,^(122m)Te, ^(125m)Te, ¹⁶⁵Tm, ¹⁶⁷Tm, ¹⁶⁸Tm, ¹⁹⁷Pt, ¹⁴³Pr, ⁵⁷Co, ⁵¹Cr,⁷⁵Se, ²⁰Tl, ⁷⁶Br and ¹⁶⁹Yb.
 21. The method of claim 15, wherein theradionuclide is ⁹⁰Y.
 22. The method of claim 15, wherein theanti-pancreatic cancer antibody or antigen-binding fragment thereof isconjugated to a radionuclide and method further comprises administeringa radiosensitizing agent to the subject before the anti-pancreaticcancer antibody or fragment is administered.
 23. A method of detectingor diagnosing pancreatic cancer comprising: a) administering to asubject an antibody construct comprising at least one chimeric,humanized or human anti-pancreatic cancer antibody or antigen-bindingfragment thereof that can bind to a linear peptide comprising the aminoacid sequence WTWNITKAYPLP (SEQ ID NO:29) or to a cyclic peptidecomprising the amino acid sequence ACPEWWGTTC (SEQ ID NO:30); and b)detecting the antibody construct bound to pancreatic cancer cells. 24.The method of claim 23, wherein the anti-pancreatic cancer antibody orantigen-binding fragment thereof binds to PanIN-1A, PanIN-1B and PanIN-2but not to pancreatitis or normal pancreatic tissue.
 25. The method ofclaim 23, wherein the anti-pancreatic cancer antibody or antigen-bindingfragment thereof is conjugated to at least one diagnostic agent.
 26. Themethod of claim 25, wherein the diagnostic agent is selected from thegroup consisting of a radionuclide, a contrast agent, a fluorescentagent, a chemiluminescent agent, a bioluminescent agent, a paramagneticion, an enzyme and a photoactive diagnostic agent.
 27. The method ofclaim 26, wherein the diagnostic agent is a radionuclide selected fromthe group consisting of ¹¹⁰In, ¹¹¹In, ¹⁷⁷Lu, ¹⁸F, ⁵²Fe, ⁶²Cu, ⁶⁴Cu,⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁸⁶Y, ⁹⁰Y, ⁸⁹Zr, ^(94m)Tc, ⁹⁴Tc, ^(99m)Tc, ¹²⁰I, ¹²³I,¹²⁴I, ¹²⁵I, ¹³¹I, ¹⁵⁴⁻¹⁵⁸Gd, ³²F, ¹¹C, ¹³N, ¹⁵O, ¹⁸⁶Re, ¹⁸⁸Re, ⁵¹Mn,^(52m)Mn, ⁵⁵Co, ⁷²As, ⁷⁵Br, ⁷⁶Br, ^(82m)Rb, ⁸³Sr, or other gamma-,beta-, or positron-emitters.
 28. The method of claim 27, wherein theradionuclide is ¹⁸F and the method further comprising PET imaging of the¹⁸F-labeled antibody or antibody fragment.
 29. The method of claim 26,wherein the paramagnetic ion is selected from the group consisting ofchromium (III), manganese (II), iron (III), iron (II), cobalt (II),nickel (II), copper (II), neodymium (III), samarium (III), ytterbium(III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III),holmium (III) and erbium (III).
 30. The method of claim 26, wherein thediagnostic agent is a fluorescent labeling compound selected from thegroup consisting of fluorescein isothiocyanate, rhodamine,phycoerytherin, phycocyanin, allophycocyanin, o-phthaldehyde andfluorescamine, or a chemiluminescent labeling compound selected from thegroup consisting of luminol, isoluminol, an aromatic acridinium ester,an imidazole, an acridinium salt and an oxalate ester, or abioluminescent compound selected from the group consisting of luciferin,luciferase and aequorin.
 31. The method of claim 23, wherein the methodis used in intraoperative, endoscopic, or intravascular procedure. 32.The method of claim 23, wherein the antibody construct is a is a DNL(dock and lock) construct comprising (i) at least one chimeric,humanized or human anti-pancreatic cancer antibody or antigen-bindingfragment thereof that can bind to a linear peptide comprising the aminoacid sequence WTWNITKAYPLP (SEQ ID NO:29) or to a cyclic peptidecomprising the amino acid sequence ACPEWWGTTC (SEQ ID NO:30); and (ii) asecond antibody or antigen-binding fragment thereof attached to the atleast one chimeric, humanized or human anti-pancreatic cancer antibodyor antigen-binding fragment thereof, wherein the second antibody bindsto an HSG or In-DTPA hapten, wherein the method further comprisesadministering to the individual a targetable construct comprising atleast one HSG or In-DTPA hapten, and wherein the targetable construct isconjugated to at least one diagnostic agent.
 33. The method of claim 23,wherein binding of the antibody construct to a sample distinguishes thepresence of pancreatic cancer from pancreatitis, normal pancreatictissue or benign pancreatic tissue in the sample.
 34. The method ofclaim 23, wherein binding of the antibody construct to a sample isdiagnostic of the presence of early stage pancreatic cancer in thesample.
 35. The method of claim 33, wherein the antibody is exposed tothe sample in vivo or in vitro.
 36. The method of claim 35, wherein thesample is a fresh serum sample and the method further comprisesperforming an organic phase extraction on the serum sample before thesample is exposed to the antibody.
 37. The method of claim 36, whereinthe organic phase is butanol.